Compositions targeting fibroblast activating proteins and methods of use thereof

Abstract

The invention discloses a compound represented by the following structural formula (I). Or a pharmaceutically acceptable salt thereof. The variables of Structural Formula (I) are described herein. The compounds of the invention can be attached to chelating groups for radionuclide binding, and thus are suitable for radioimaging and / or radiotherapy applications, for example, the disclosed compounds can be radiolabeled with positron emitters such as 18F, 68Ga or 64Cu, and used for positron emission tomography (PET). Alternatively, the compounds can be radiolabeled with an alpha particle emitter such as 225 Ac, a beta particle emitter such as 67 Cu or 177 Lu or Auger electron emitters (e.g., 111 In, 67 Ga, 99 mTc, 195 mPt, 125 I, and 123 I). The compounds may also be attached to a cytotoxic agent for targeted delivery of the cytotoxic agent to a tumor, such as conjugated to gemcitabine or doxycycline or venom. Likewise, the compound may be conjugated to a compound having a physiological action, such as a TLR agonist, to stimulate the immune response of the receptor.

Description

Related applications

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 627,309, filed January 31, 2024, and U.S. Provisional Application Serial No. 63 / 529,522, filed July 28, 2023, the entire teachings of which are incorporated herein by reference. Technical Field

[0002] This technology relates to targeted imaging and therapeutic agents, and more specifically, to compounds that can be used for the diagnosis and treatment of diseases. For example, the compositions described herein can be used as radiopharmaceuticals, or conjugated with optical dyes or fluorophores, or as drug / toxin conjugates for the diagnosis and treatment of cancers and fibrotic diseases in tissues. Background Technology

[0003] Fibroblast activation protein α (“FAP”) is a 170 kDa type II membrane-bound enzyme exhibiting serine protease activity. Soluble forms of FAP, lacking both the full-length intracellular and transmembrane sequences, are found in plasma. Other common names for FAP include prolyl endopeptidase and seprase.

[0004] FAP is one of several members of the S9B prolyl oligopeptidase subfamily, which includes proteins such as DPP4, DPP8, and DPP9. FAP substrates include neuropeptide Y, peptide YY, substrate P, B-type natriuretic peptide, fibroblast growth factor 21 (FGF-21), α2 antifibrinolytic enzyme, and denaturing collagen I and III.

[0005] FAP is actively expressed in tissues undergoing wound healing and remodeling, but is not expressed or is expressed at very low levels in healthy, mature tissues. Tumors are localized areas of host tissue damage, actively involved in remodeling the local vascular system and endothelium, as well as several other phenomena. They help hide tumors from immune surveillance and promote wound healing, a microenvironment that allows tumor cell proliferation. Therefore, FAP expression is associated with tumorigenic tissue regions, particularly the tumor stroma, thus providing an excellent molecular target for the diagnosis and treatment of various cancers. Its expression has been confirmed in many cancers, such as pancreatic cancer, liver cancer, gallbladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, kidney cancer, melanoma, and many other deadly and aggressive tumors and cancer types. FAP expression has also been detected in fibrotic tissue and can be used as a biomarker for a wide range of clinical conditions, including systemic fibrotic diseases such as systemic sclerosis (SSc), sclerosing skin graft-versus-host disease, and renal systemic fibrosis, as well as multiple organ-specific conditions including radiation-induced fibrosis and fibrosis of the heart, lungs, liver (such as NAFLD: non-alcoholic fatty liver disease and NASH: non-alcoholic steatohepatitis), and kidneys.

[0006] Although some researchers have explored FAP as a cancer target, its pharmacokinetic limitations have hampered the development of diagnostic or even therapeutic agents. To date, FAP has proven to be a challenging target due to its low and limited expression and the limited residence time of existing compounds in vivo. Improved FAP binders exhibiting better binding kinetics and biodistribution are needed to provide a foundation for improved FAP-targeting diagnostic and therapeutic agents—compounds capable of accumulating at higher levels in tumors and not being unacceptably taken up in normal non-target tissues and organs. Summary of the Invention

[0007] FAP-targeted therapy is emerging as a potential approach for treating various cancers in which FAP is expressed in the tumor microenvironment and / or on the tumor cells themselves. Several FAP-targeting molecules, such as FAPI-04, FAPI-46, FAP-2286, and PNT6555, have been investigated in preclinical studies and early-stage clinical trials. FAP-targeted therapy has shown some preliminary efficacy in humans; however, the relatively short tumor retention time is a significant limitation. Current FAP-targeting compounds are cleared from tumor tissue relatively quickly, limiting the radiation dose absorbed by the tumor. Despite efforts to improve tumor retention of FAP-targeted radiotracers, progress has been limited.

[0008] This article discloses a series of novel compounds that bind to the extracellular domain of FAP. Unlike existing compounds, the disclosed compounds exhibit unique chemical structures that significantly enhance their affinity for FAP (see Examples 2 and 3), thereby improving tumor retention.

[0009] The compounds disclosed herein can attach to chelating groups for the binding of radionuclides, and are therefore suitable for radiographic imaging and / or radiotherapy applications, for example, the disclosed compounds can be used with positron emitters such as... 18 F, 68 Ga or 64 Cu was radiolabeled and used in positron emission tomography (PET) (see Example 4). Alternatively, the compound could be radiolabeled using an alpha particle emitter such as... 225 Ac and β particle emitters, such as 67 Cu or 177 Lu or Auger electron emitters (e.g.) 111 In、 67 Ga、 99 mTc, 195 mPt, 125 I and 123 I) Radiolabeling. The compounds can also be conjugated to cytotoxic agents for targeted delivery to tumors, for example, conjugated to gemcitabine or doxycycline or venom. Similarly, the compounds can be conjugated to compounds with physiological effects, such as TLR agonists, to stimulate an immune response in the receptor. The disclosed compounds have the advantage of a surprisingly high affinity for FAP. Many of these compounds also have the effect of binding to albumin / prolonging circulation residence time, but it is precisely their high affinity for FAP that is considered to be the reason for their significant residence time in tumors. The increased circulation residence time has the effect of increasing target load while reducing compound accumulation in non-target tissues (see Example 6). Therefore, the favorable binding kinetics of the compounds disclosed herein reduce the “elution” effect (i.e., low residence time) of prior art FAP-targeting compounds.

[0010] One embodiment of the present invention is a compound represented by the following structural formula (I): Or its pharmaceutically acceptable salt, wherein: n is 0 or 1; A is NH, O, S, or CR. 6 R 7 ; B comprises a branched, unbranched, or cyclic aliphatic group of up to 30 carbon atoms, optionally interrupted by a peptide chain of up to 10 heteroatoms or up to 20 amino acid residues (e.g., 3-20 or 3-15 carbon atoms, optionally interrupted by up to 6 heteroatoms or up to 5 amino acid residues), wherein B is optionally substituted by 1-5 groups selected from: F, Cl, Br, I, =O, OR 6 OCOR 6 COOR 6 CN, =NR 6 NR 6 R 7 =S and SR 6 The condition is that B contains at least 3 atoms in the chain between group D and group A; D is selected from: OPO3H2, PO3H2, OSO3H, SO3H and COOH or their C1-C4 alkyl esters; X is O or S; R 1 It is a chelating group, an optical dye or fluorophore, a cytotoxic agent, an immunostimulant, or optionally composed of one or more R groups. 5 The group indicated is the benzoyl group that has been substituted; R 3 It is a C1-C8 alkyl or a C1-C4 aralkyl, wherein: The aryl moiety of alkyl and aralkyl groups is optionally and independently surrounded by F, Cl, Br, I, branched, unbranched, or cyclic C1-C6 aliphatic groups, OR 6 OCOR 6 COOR 6 CHO, COR 6 CH2OR 6 NR 6 R 7 CH2NR 6 R 7 SR 6 =O, =S and =NH substitution; R 4 It is either CN or B(OH)2; Each R 5 Independently selected from halogen, cyano, halomethyl, N + (CH3)3W - W - It is a pharmaceutically acceptable anion; R 6 and R 7 Independently selected from: H or C1-C6 alkyl; and R 8 It is C 1-4 Alkyl, and R9 Selected from H and C1-C4 alkyl groups, or R 8 and R 9 Together with its middle carbon atom, it forms a C3-C6 cycloalkyl group.

[0011] Another embodiment of the invention is a pharmaceutical composition comprising: i) a compound disclosed herein or a pharmaceutically acceptable salt thereof; and ii) a pharmaceutically acceptable carrier or diluent. For compounds containing a chelating group, the chelating group is preferably chelated with a radionuclide.

[0012] Another embodiment of the invention is a method for treating a subject with diseased tissue expressing fibroblast activation protein α. In one aspect, the diseased tissue may be cancerous or fibrotic tissue. The method includes administering to the subject an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt. Preferably, the compound used for treatment includes a cytotoxic agent, such as a chelating group having a radionuclide that emits β, α, Auger, or other cytotoxic rays capable of killing diseased tissue.

[0013] Another embodiment of the present invention is a method for imaging a region in a subject having or suspected of having diseased or fibrotic tissue expressing fibroblast activation protein α, comprising: a. Administering to a subject a diagnostically effective amount of the compound disclosed herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein; b. Exposing a region of the subject to an imaging device, the region suspected of containing diseased tissue; and c. Obtain images of diseased tissue in the region.

[0014] Preferably, the compound used for imaging comprises a radionuclide or other chelating group that emits gamma rays or positrons and is otherwise detectable by irradiation. In another aspect, the compound comprises an optical dye or fluorophore that can detect its emission.

[0015] Another embodiment of the present invention is a method for tumor imaging. The method includes: a. Administer to a subject the compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount that effectively contacts and binds to the tumor and / or surrounding tissue; b. Irradiate the tumor and / or surrounding tissues at wavelengths absorbed by the bound compound; c. And the detection of signals from irradiated binding compounds, thereby imaging the tumor and / or surrounding tissues.

[0016] Preferably, the compound used for imaging contains a chelating group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable irradiation.

[0017] Another embodiment of the present invention is a method for treating diseased tissue. The method includes: a. Administer to a subject the compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount that effectively contacts and binds to diseased tissue; b. Using a compound as a reference, the region bound to the compound is irradiated with one or more doses of external irradiation. Therefore, irradiation therapy is used to treat diseased tissues.

[0018] Preferably, the compound used as a reference contains a chelating group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable irradiation.

[0019] In yet another embodiment of the invention is a method for treating diseased tissue. The method includes: a. Administer to a subject the compound disclosed herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount that effectively contacts and binds to diseased tissue; b. Use compounds as a guideline for surgical application to remove areas of diseased tissue. This allows for the removal of the diseased tissue.

[0020] Preferably, the compound used as a reference contains a chelating group having a radionuclide that emits gamma rays or positrons, or an optical dye or fluorophore, or other detectable irradiation. Attached Figure Description

[0021] Figure 1 This shows the organ biodistribution of [Lu-177]RTX-1371R in BALB / C nude mice previously transplanted with U-87 / MG cells.

[0022] Figure 2 This shows the organ biodistribution of [Lu-177]RTX-1391R in BALB / C nude mice previously transplanted with U-87 / MG cells.

[0023] Figure 3 This shows the organ biodistribution of [Lu-177]RTX-1392R in BALB / C nude mice previously transplanted with U-87 / MG cells.

[0024] Figure 4This shows the organ biodistribution of [Lu-177]RTX-1356R and [Lu-177]RTX-1391R in BALB / C nude mice previously transplanted with U-87 / MG cells.

[0025] Figure 5 This shows the organ biodistribution of [Lu-177]RTX-1357R and [Lu-177]RTX-1392R in BALB / C nude mice previously transplanted with U-87 / MG cells.

[0026] Figure 6 This shows the organ biodistribution of [Lu-177]RTX-1359R and [Lu-177]RTX-1371R in BALB / C nude mice previously transplanted with U-87 / MG cells.

[0027] Figure 7 This shows the organ biodistribution of [Lu-177]RTX-1427R and [Lu-177]RTX-1411R in BALB / C nude mice previously transplanted with U-87 / MG cells.

[0028] Figure 8 This shows the organ biodistribution of [Lu-177]RTX-1418R in BALB / C nude mice previously transplanted with U-87 / MG cells.

[0029] Figure 9 This shows the organ biodistribution of [Lu-177]RTX-1386S in BALB / C nude mice previously transplanted with U-87 / MG cells.

[0030] Figure 10 This shows the organ biodistribution of [ZW800-1]RTX-1384S in female Nu / J or NCr-Foxn1 nude mice that had previously received U-87 / MG cells.

[0031] Figure 11 This shows the organ biodistribution of [ZW800-1]RTX-1384S in female Nu / J or NCr-Foxn1 nude mice previously transplanted with U-87 / MG cells, normalized to muscle.

[0032] Figure 12 This study demonstrates the efficacy of different doses of [Ac-225]RTX-1359R in female Nu / J mice previously transplanted with U-87 / MG cells.

[0033] Figure 13The percentage of female Nu / J mice previously transplanted with U-87 / MG cells survived after treatment with [Ac-225]RTX-1359R.

[0034] Figure 14 This study demonstrates the efficacy of different doses of [Ac-225]RTX-1392R in female Nu / J mice previously transplanted with U-87 / MG cells.

[0035] Figure 15 The percentage of female Nu / J mice previously transplanted with U-87 / MG cells survived after treatment with [Ac-225]RTX-1392R.

[0036] Figure 16 This study compares the efficacy of [Ac-225]RTX-1392R and [Ac-225]RTX-1411R in female Nu / J mice previously transplanted with U-87 / MG cells.

[0037] Figure 17 This study compares the survival percentages of female Nu / J mice previously transplanted with U-87 / MG cells after treatment with [Ac-225]RTX-1392R or [Ac-225]RTX1411R. Detailed Implementation

[0038] This document discloses a series of compounds that bind with high affinity to the extracellular domain of FAP and are capable of delivering payloads to FAP-expressing tissues. The compounds of this invention are described below.

[0039] The first embodiment of the present invention is a compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof, wherein the variables are the same as those described above with respect to structural formula (I).

[0040] A second embodiment of the invention is a compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof, wherein B is independently a branched or unbranched aliphatic group of 3 to 20 carbon atoms, said carbon atoms optionally being interrupted by a peptide chain of up to 10 heteroatoms or up to 5 amino acid residues, said aliphatic group optionally being F, Cl, Br, I, =O, OR 6 OCOR 6 COOR 6 CN, =NR 6 NR 6 R 7 =S or SR 6 Replace them, the remaining variables are the same as those described above for structure (I).

[0041] The third embodiment of the present invention is a compound represented by structural formula (II): (II); Or its pharmaceutically acceptable salt, wherein m is an integer from 0 to 12; o is 0 or 1; and R 2 It is H or C1-C4 alkyl; and the remaining variables are as described in the first or second embodiment.

[0042] The fourth embodiment of the present invention is a compound represented by structural formula (III): (III); Or a pharmaceutically acceptable salt thereof; wherein the variables are as described in the third embodiment.

[0043] The fifth embodiment of the present invention is a compound represented by structural formula (IV): (IV); Or a pharmaceutically acceptable salt thereof; wherein the variables are as described in the third embodiment.

[0044] The sixth embodiment of the present invention is a compound represented by structural formula (V): (V); Or a pharmaceutically acceptable salt thereof; wherein the variables are as described in the third embodiment.

[0045] The seventh embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 3 It can be optionally reacted with F, Cl, Br, I or C 1-4 Alkyl-substituted C 1-8 Alkyl or C 1-4 Aryl alkyl groups, and other variables as described in the first, second, or third embodiments.

[0046] The eighth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 3 It is optional to be I or C 1-4 Alkyl-substituted C 1-8 Alkyl or C 1-4 Aryl alkyl groups, and other variables as described in the first, second, third, or seventh embodiments.

[0047] The ninth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 3It is a C1-C8 alkyl or C1-C4 aralkyl that is optionally substituted with a C1-C4 alkyl group, and the remaining variables are as described in the first, second or third, seventh or eighth embodiments.

[0048] The tenth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 3 It is methyl, (4-isobutylphenyl)methyl, (4-isobutylphenyl)propyl, (4-iodophenyl)methyl or (4-iodophenyl)propyl, and the remaining variables are as described in the first, second, third, seventh or eighth embodiments.

[0049] The eleventh embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 3 It is methyl, (4-isobutylphenyl)methyl and (4-isobutylphenyl)propyl, and the remaining variables are as described in the first, second, third, seventh, eighth, ninth or tenth embodiments.

[0050] The twelfth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 8 It is methyl and R 9 H is the variable, and the remaining variables are as described in the first, second, third, seventh, eighth, ninth, tenth, or eleventh implementation schemes.

[0051] The thirteenth embodiment of the invention is a compound represented by structural formula (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein o is 1 and m is 3 to 12, and the remaining variables are as described in the third, seventh, eighth, ninth, tenth, eleventh or twelfth embodiments.

[0052] The fourteenth embodiment of the invention is a compound represented by structural formula (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein m is 8, and the remaining variables are as described in the thirteenth embodiment.

[0053] The fifteenth embodiment of the invention is a compound represented by structural formula (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein o is 0, and the remaining variables are as described in the third, seventh, eighth, ninth, tenth, eleventh or twelfth embodiments.

[0054] The sixteenth embodiment of the invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein n is 1; and the remaining variables are as described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth embodiments.

[0055] The seventeenth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 1 It is a fluorophore or optical dye; and the remaining variables are as described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, or sixteenth embodiments. In one aspect, the fluorophore is The optical dyes are carbocyanine, indole carbocyanine, oxycarbocyanine, sulfur carbocyanine, cyanine, polyacetylenes, coumarin, rhodamine, xanthan, fluorescein, boron dipyrrole methylene (BODIPY), VivoTag-680, VivoTag-S750, AlexaFluor dyes (e.g., AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790) and DylightFluor dyes.

[0056] The eighteenth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 1 It is a chelating group, which is a residue of the chelating agent; and the remaining variables are as described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth or sixteenth embodiments. Suitable chelating agents and the residues of chelating agents are described below.

[0057] The nineteenth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 1 It is optionally controlled by one or more R 5 The group represented is the substituted benzoyl group; each R 5 Independently selected from halogen, cyano, halomethyl or N + (CH3)3W - And W - It is a pharmaceutically acceptable anion; and the remaining variables are as described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, or sixteenth embodiments. In one aspect, R5 The halogen group it represents is 18 F.

[0058] The twentieth embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein each R 5 Independently selected from fluorine, cyano, trifluoromethyl or N + (CH3)3W - And the remaining variables are as described in the nineteenth implementation scheme. In one aspect, R 5 The fluorine group it represents is F. 18 .

[0059] The twenty-first embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 2 It is H and R 4 It is B(OH)2; and the remaining variables are as described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth or twentieth implementation schemes.

[0060] The twenty-second embodiment of the present invention is a compound represented by structural formula (I), (II), (III), (IV) or (V), or a pharmaceutically acceptable salt thereof, wherein R 2 It is H and R 4 It is CN; and the remaining variables are as described in the first, second, third, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth or twentieth implementation schemes.

[0061] The invention also includes compounds whose preparation methods are described in the examples and shown in the accompanying drawings, including both pharmaceutically acceptable salts and neutral forms. For those compounds containing chelating groups, chelation with radionuclides is also included in this invention.

[0062] The present invention also includes the compounds shown below, including both pharmaceutically acceptable salts and neutral forms thereof. Chelation with radionuclides is also included in the present invention.

[0063] RTX-1371R RTX-1384S RTX-1391R RTX-1392R RTX-1400R RTX-1401R RTX-1402R RTX-1407S RTX-1409R The nomenclature method of adding an isotope before the compound name indicates that the isotope is chelated with the compound's chelating group. For example, "[68Ga]RTX-1371R" refers to RTX-1371R, where its chelating group is chelated with... 68Ga chelation. Exemplary compounds (and their chelating groups) of the present invention include [68Ga]RTX-1371R; [68Ga]RTX-1391R; [68Ga]RTX-1392R; [68Ga]RTX-1411R; [212Pb]RTX-1371R; [212Pb]RTX-1391R; [212Pb]RTX-1392R; [212Pb]RTX-1411R; [90Y]RTX-1371R; [90Y]RTX-1391R; [90Y]RTX-1392R; [90Y]RTX-1411R; [117Sn]RTX-1371R; [117Sn]RTX-1391R; [ 117Sn]RTX-1392R; [117Sn]RTX-1411R; [186Re]RTX-1371R; [186Re]RTX-1391R; [186Re]RTX-1392R; [186Re]RTX-1411R; [188Re]RTX-1371R; [188R e]RTX-1391R;[188Re]RTX-1392R;[188Re]RTX-1411R;[169Er]RTX-1371R;[169Er]RTX-1391R;[169Er]RTX-1392R;[169Er]RTX-1411R;[153Sm]RTX -1371R;[153Sm]RTX-1391R;[153Sm]RTX-1392R;[153Sm]RTX-1411R;[223Ra]RTX-1371R;[223Ra]RTX-1391R;[223Ra]RTX-1392R;[223Ra]RTX-141 [161Tb]RTX-1371R; [161Tb]RTX-1391R; [161Tb]RTX-1392R; [161Tb] ]RTX-1411R;[213Bi]RTX-1371R;[213Bi]RTX-1391R;[213Bi]RTX-1392R;[213Bi]RTX-1411R;[166Ho]RTX-1371R;[166Ho]RTX-1391R;[166Ho]RTX -1392R;[166Ho]RTX-1411R;[149Tb]RTX-1371R;[149Tb]RTX-1391R;[149Tb]RTX-1392R;[149Tb]RTX-1411R;[47Sc]RTX-1371R;[47Sc]RTX-1392R; [47Sc]RTX-1411R; [227Th]RTX-1371R; [227Th]RTX-1391R; [227Th]RTX-1392R; [227Th]RTX-1411R; [177Lu]RTX-1371R; [ 177Lu]RTX-1391R; [177Lu]RTX-1392R; [177Lu]RTX-1411R; [225Ac]RTX-1371R; [225Ac]RTX-1391R; [225Ac]RTX-1392R; and [225Ac]RTX-1411R. ;

[0064] Compounds containing Macropa chelating agents are typically chelated with 225Ac. Therefore, another compound (and its chelating group) of the present invention includes [Ac225]RTX-1400R, [Ac225]RTX-1401R, [Ac225]RTX-1402R, [Ac225]RTX-1407S, [Ac225]RTX-1413R, [Ac225]RTX-1414R, [Ac225]RTX-1415R, [134Ce]RTX-1400R, [134Ce]RTX-1401R, [134Ce]RTX-1402R, [134Ce]RTX-1407S, [134Ce]RTX-1413R, [134Ce]RTX-1414R and [134Ce]RTX-1415R.

[0065] "Aliphatic" refers to a saturated or unsaturated straight-chain or branched monovalent or divalent hydrocarbon group. Unless otherwise specified, aliphatic groups typically have 1 to 10 carbon atoms. "Alkyl" refers to a saturated aliphatic straight-chain or branched monovalent aliphatic group. Unless otherwise specified, alkyl groups typically have 1 to 10 carbon atoms (C1 to C2). 1-10 Alkyl), or alternatively, 1 to 6 carbon atoms (C 1-3 Alkyl group (i.e., 1, 2 or 3).

[0066] "Cyclic aliphatic" refers to saturated or unsaturated monovalent or divalent cyclic hydrocarbon rings. Unless otherwise stated, cyclic aliphatic hydrocarbons have 3 to 8 ring carbon atoms (C6C6). 3-8 Cycloalkyl. “Cycloalkyl” means saturated aliphatic cyclic aliphatic. Unless otherwise stated, cycloalkyl has 3 to 8 cyclic carbon atoms.

[0067] "Aryl", alone or in part or more, such as "aralkyl", is a carbocyclic aromatic group, such as phenyl or naphthyl.

[0068] "Aryl group" refers to an alkyl group that has been substituted with an aryl group. "C1-C x"Aryl group" refers to an aryl group whose alkyl part contains 1 to x carbon atoms.

[0069] Compounds having one or more chiral centers can exist in various stereoisomeric forms, meaning each chiral center can have... R or S Configuration, or a mixture of both. Stereoisomers are compounds that differ only in their spatial arrangement. Stereoisomers include all diastereomeric and enantiomeric forms of a compound. Enantiomers are stereoisomers that are not superimposed mirror images of each other. Diastereomers are stereoisomers with two or more chiral centers that are not identical and are not mirror images of each other.

[0070] In compounds having one or more chiral centers, the stereochemical configuration of the chiral center is determined by its chemical name (e.g., where the configuration is indicated in the chemical name by "..."). R "or" S When described by a symbol (e.g., configuration represented by a wedge bond), the enrichment of the indicator configuration relative to the opposite configuration is greater than 50%, 60%, 70%, 80%, 90%, 99%, or 99.9%. "Enrichment of the indicator configuration relative to the opposite configuration" is a molar percentage, determined by dividing the number of compounds with the indicator stereochemical configuration at the chiral center by the total number of all compounds in the mixture having the same or opposite stereochemical configurations.

[0071] When a disclosed compound having a chiral center is described by a structure that does not show a configuration at that chiral center, the structure means that it encompasses the chiral center having S Compounds with this configuration, having at this chiral center R Compounds with this configuration or having at this chiral center R and S Compounds with mixed configurations. When a publicly known compound with a chiral center is described by its chemical name, without using "..." S "or" R When referring to the configuration of the chiral center, the name means to encompass the chiral center having S The compound with this configuration, the chiral center has R The configuration of the compound or the chiral center has R and S Compounds with mixed configurations.

[0072] The targeted FAP compounds of the present invention are useful imaging agents for diagnostic applications. For example, these can be conjugated with various metals for magnetic resonance imaging applications, or conjugated with optical dyes or fluorophores, or other detectable components (i.e., dyes, quantum dots, etc.) for histochemical and luminescence imaging applications. Similarly, these compounds can be radiolabeled and used for nuclear medicine applications. Radionuclides that can be used for imaging applications are referred to herein as "imaging radionuclides." Non-limiting examples of imaging radionuclides include… 18 F, 64 Cu or 68 Ga, which is suitable for PET imaging applications, and 67 Cu or 177 Lu, which is typically used to treat nucleotides, is also applicable to SPECT imaging applications.

[0073] The targeted FAP compounds of the present invention are useful therapeutic compounds. Such therapeutic compounds include the targeted FAP compounds of the present invention having suitable therapeutic portions. The targeted FAP compounds can be separated from the therapeutic portions via covalent links. The spacing between these (based on successive atomic counts) can be from about 4 atoms to about 100 atoms. Furthermore, by including additional targeting structures on the compound, the pharmacokinetics of the compound can be altered. For example, using a blood-targeting portion can increase circulation residence time, which has the effect of increasing tumor perfusion and load, while reducing the accumulation of radiotherapy compounds in non-target tissues. See, for example, U.S. Patent 11,285,277, which describes a trifunctional (“Trillium”) compound having a tumor-targeting domain, a blood protein-binding domain, and a third domain constituting a cell-killing or cell-growth-inhibiting therapeutic agent. In the presently preferred embodiment, the binding FAP compounds of the present invention are capable of binding to a Trillium scaffold via covalent bonds to constitute the tumor-targeting domain of a Trillium agent of such a targeted FAP. Exemplary constructs include pharmaceutical conjugates having toxins, venoms, metabolites, or chemotherapeutic agents, as well as radiotherapy compounds having alpha-emitting radionuclides, beta-emitting radionuclides, or Auger electron-emitting radionuclides, or emitting radiation spectra upon decay (including positron emission, which is also applicable to diagnostic purposes).

[0074] For radiotherapy, targeted FAP compounds are conjugated with chelating agents, the choice of which is based on its suitability to contain appropriate therapeutic radionuclides. "Therapeutic radionuclides" are radionuclides that can be used for therapeutic purposes, such as treating cancer or fibrotic tissue due to their radioactive emission, and that exhibit cytotoxic effects on the target tissue (i.e., cancer and tumor microenvironments expressing FAP, malignant tumors, and fibrotic cells). Although targeted radiotherapy using macrocyclic complexes of radionuclides has been practiced for some time, currently used macrocycles (e.g., DOTA) typically form complexes with many therapeutic radionuclide metals, such as actinium, radium, bismuth, astatine, lutetium, and lead isotopes. The instability of many known macrocyclic compounds can cause the radionuclide to dissociate from the macrocycle, resulting in a lack of selective delivery to the intended target tissue, which can also lead to toxicity to non-target tissues. Alpha-emitting radionuclides, such as… 225 Ac, which can provide greater cytotoxic effects, is therefore considered more effective for treatment than beta-emitting radionuclides. However, this toxin requires chelating agents to increase the retention of chelated metals. U.S. Patent 11,279,698 (see also PCT / US2018 / 025488 and PCT / US2019 / 062479) describes a novel chelating agent (“Macropa”) and its role as a chelating agent. 225 Ac chelators are used, including as components of Trillium PK-modified targeted radiotherapy agents. The ratio of tumor activity to renal activity is 1 or higher, and can persist for up to approximately 36 hours after radiotherapy administration, and in the case of 225Ac Trillium-based agents, for 72 hours or even 128 hours or longer, to maximize the therapeutic effect of irradiation on the target tissue.

[0075] Therefore, an exemplary preferred Trillium compound targeting FAP will have a chelating agent in its third (untargeted) domain. Macropa is currently targeted... 225 Preferred chelating agents for the Trillium compound in Ac-FAP. (See also PCT / CA2021 / 050226).

[0076] The radionuclides that can be used with the compounds disclosed herein depend on the application, the type of radiation required, and the half-life, as will be apparent to those skilled in the art. Exemplary radionuclides include: 177 Lu、 175 Lu、 45 Sc、 47 Sc、 64 Cu、 67 Cu、 68Cu、 66 Ga、 67 Ga、 68 Ga、 69 Ga、 71 Ga、 90 Y、 89 Y、 86 Y、 89 Zr、 90 Y、 99m Tc, 111 In、 113 In、 115 In、 117 Sn、 153 Sm、 139 La、 134 Ce、 136 Ce、 138 Ce、 140 Ce、 142 Ce、 151 Eu、 153 Eu、 152 Dy、 149 Tb, 159 Tb, 154 Gd, 155 Gd, 156 Gd, 157 Gd, 158 Gd, 160 Gd, 161 Tb, 166 Ho、 169 Er、 188 Re、 186 Re、 213 Bi、 211 At、 217 At、 227 Th、 226 Th、 225 Ac、 233 Ra、 152 Dy、 213 Bi、 212 Bi、 211 Bi、 203 Pb, 212 Pb, 223 Ra、 255 Fm and uranium-230. Based on their decay properties, the radionuclides in any of the embodiments described herein can be either therapeutic or diagnostic radionuclides. Currently, preferred alpha-emitting radionuclides for therapeutic applications include... 225 Ac、 233 Ra and 212 Pb. Preferred β-emitting radionuclides for therapeutic applications include177 Lu, 90 Y, and 67 Cu.

[0077] Chelating groups and aza-macrocyclic polycarboxylic acids that can be used in this technology include, and refer to, groups capable of chelating, binding, or otherwise delivering radionuclides to therapeutic or diagnostic targets. A chelating group is a residue following the reaction of a chelating agent with a nucleophilic group in a compound to form a targeted divalent radiopharmaceutical or radiodiagnostic agent capable of binding and delivering radionuclides. In the case of the disclosed compounds, the reactive group is a side-chain amine of the lysine acyl group in the penultimate precursor, which reacts with the chelating agent to form the disclosed compound.Examples of chelating agents include, but are not limited to, substituted or unsubstituted members covalently conjugated with the following groups: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), p-SCN-Bn-NOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS), PIP-DOTA, diethylenetriaminepentaacetic acid (DTPA), PIP-DTPA, AZEP-DTPA, ethylenediaminetetraacetic acid (EDTA), triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid (TTHA), 7-[2] [-(bis(carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraazacyclododecane-1-yl-acetic acid (DEPA), 2,2',2''-(10-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanophenyl)pentyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (3p-C-DEPA-NCS), NETA, {4-carboxymethyl-7-[2-(carboxymethylamino)-ethyl]-perhydro-1,4,7-triazacyclononane-1-yl}-acetic acid (NPTA), diacetylpyridinium bis(benzoylhydrazone), 1,4,7,10,13,16-hexaazacyclooctadecane N ,N',N'',N''',N'''',N'''''-Hexaacetic acid (HEHA), Octadecate terephthalamide ligand, 2,2'-(4-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanophenyl)pentyl)-10-(2-(bis(carboxymethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diaacetic acid, N,N'-bis[(6-carboxy-2-pyridine)methyl]-4,13-diaza-18-crown-6 (H2macropa), 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl) -4-Isothiocyanopyridinecarboxylic acid (macropa-NCS), 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-isocyanopyridinecarboxylic acid (macropa-NCO), 3,9-carboxymethyl-6-(2-methoxy-5-isothiocyanophenyl)carboxymethyl-3,6,9,15-tetraazabicyclo-[9.3.1]pentadecane-1(15),11,13-triene and 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetamide (TCMC or DOTAM).In the currently preferred embodiment, the chelating agent is a macrocyclic residue of a polyazapolycarboxylic acid, such as MacropaNCS or NCO-Macropa. In another aspect, the chelating agent is a ferrophilic residue; in one aspect, ... 225 Ac is a radionuclide targeting Macropa NCS or NCO-Macropa. In another aspect, the chelating agent is a residue of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOA, or DOTA. In another embodiment, the chelating agent is a sarcophagene chelating agent. On the other hand, the chelating agent is... 68 Ga-chelated residues of p-SCN-Bn-DOTA, p-SCN-Bn-NOTA, NOTA, or DOTA.

[0078] As described above, the disclosed compounds or pharmaceutically acceptable salts thereof may contain one or more radionuclides suitable for use as radioactive imaging agents. Imaging methods include positron emission tomography (PET) or single-photon emission computed tomography (SPECT). Therefore, in another aspect, the present invention provides therapeutic applications, namely, administering to a subject suffering from cancer, tumor, or fibrosis an effective amount of the disclosed compound (or a pharmaceutically acceptable salt thereof) having a chelating agent, which is complexed with an imaging radionuclide for the imaging application, and methods of administering an effective amount of the compound complexed with a therapeutic radionuclide for treatment. Exemplary cancers that can be imaged and / or treated using the disclosed compounds or pharmaceutically acceptable salts thereof include pancreatic cancer, liver cancer, gallbladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, kidney cancer, prostate cancer, colorectal cancer, soft tissue sarcoma, osteosarcoma, or melanoma.

[0079] "Subject" is a mammal that requires medical treatment or diagnosis, preferably a human, but may also be an animal that requires veterinary treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.) and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0080] The disclosed compounds or their pharmaceutically acceptable salts (including chelates with radionuclides) or pharmaceutical compositions thereof may be administered orally or via a parenteral route (usually by injection or infusion). “Parenteral route” means a mode of administration other than enteral and local administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, and intrasternal injections and infusions.

[0081] The “effective amount” of the disclosed compound or its pharmaceutically acceptable salt (including chelation with radionuclides) refers to the amount of a therapeutic agent used alone or in combination with other therapies that provides therapeutic benefit in treating or preventing disease, improves overall therapy, reduces or avoids disease symptoms or causes, or enhances or synergizes with another therapeutic agent. Regarding imaging, the “diagnostic effective amount” refers to the amount capable of producing useful images for diagnosis, such as the presence of a tumor. The “effective amount” of the disclosed compound or its pharmaceutically acceptable salt is determined by a physician based on patient-specific parameters such as age, weight, sex, and disease severity. The preferred dosage is 0.0001 mg / kg to 100 mg / kg body weight.

[0082] Corresponding to the type of administration, the drug is appropriately formulated, for example, in the form of a solution or suspension, a simple tablet or sugar-coated pill, a hard or soft gelatin capsule, a suppository, an ovule, or an injectable preparation, which is prepared according to the conventional Galen method.

[0083] When using infusion or injection solutions, aqueous solutions or suspensions are preferred, which can be produced prior to use, for example, from lyophilized formulations containing the active substance itself or together with a carrier (such as mannitol, lactose, glucose, albumin, etc.). The pre-prepared solution is sterilized and, where appropriate, mixed with excipients, such as preservatives, stabilizers, emulsifiers, solubilizers, buffers, and / or salts, to adjust the osmotic pressure. Sterilization can be achieved through aseptic filtration using a filter with small pore sizes, and, depending on the composition, lyophilization may be performed where appropriate. A small amount of antibiotics may also be added to ensure maintenance of sterility.

[0084] According to another aspect, a pharmaceutical composition suitable for in vivo imaging and / or radiotherapy of target tissues is provided. A suitable pharmaceutical composition may comprise a radioactive imaging agent together with a pharmaceutically acceptable radioactive carrier having a radionuclide as an element (i.e., 18 F), or diagnostic radioactive metal chelate complexes (e.g., with...). 64 Cu or 68 (Ga), or radioactive therapeutic agents, which are radioactive metal chelate complexes in sufficient quantity to bind to target tissue. Radioactive carriers should be suitable for injection or aspiration, such as human serum albumin; aqueous buffer solutions, for example, tris(hydroxymethyl)aminomethane (and its salts), phosphates, citrates, bicarbonates, etc.; sterile saline solutions; and balanced ion solutions containing chlorides and / or bicarbonates, or normal plasma containing calcium, potassium, sodium, and magnesium.

[0085] The concentration of the radiopharmaceutical in the radiopharmaceutical carrier should be sufficient to provide reasonable binding to the target tissue, such as about 4% to 20% ID / gram. For example, when using an aqueous solution, the human dose can be in the activity range of about 1.0 to 500 mCurie. However, the actual dose administered to the patient for imaging or therapeutic purposes is determined by the physician administering the treatment. The administration of imaging or therapeutic agents should allow them to remain in the patient for about 1 hour to 10 days, but longer and shorter periods are acceptable. Therefore, convenient ampoules containing 1 to 10 mL of aqueous solution can be prepared.

[0086] Imaging can be performed in a normal manner, for example, by injecting a sufficient amount of the imaging composition to provide adequate imaging, followed by scanning with a suitable imaging or scanning instrument (such as a tomography scanner or gamma camera). In some embodiments, a method of imaging a region in a patient includes the steps of: (i) administering a diagnostically effective amount of a compound complexed with a radionuclide to the patient; exposing a region of the patient to a scanning device; and (ii) obtaining an image of the patient's region. Thus, the present invention provides a method for obtaining images of a mammalian subject after administration of the compound. Similarly, imaging can be performed after administration of a therapeutic agent or a cycle of radiotherapy to assess efficacy. Therefore, obtaining images after administration of a radiotherapy agent can occur after approximately 1 hour, approximately 4 hours, approximately 9 hours, approximately 12 hours, approximately 16 hours, approximately 20 hours, approximately 24 hours, approximately 36 hours, approximately 48 hours, approximately 72 hours, approximately 96 hours, approximately one week, approximately two weeks, approximately four weeks, or after completion of a treatment cycle. Therefore, in some embodiments, a method for imaging tissue (such as tumor tissue expressing FAP) is provided, which includes contacting the tissue with a complex synthesized by contacting an imaging radionuclide with a disclosed compound.

[0087] According to another aspect, the disclosed compound or a pharmaceutically acceptable salt complex thereof may contain one or more radionuclides suitable for use as a radioimaging agent in the field of image-guided radiotherapy (IGRT). As described in U.S. Patent No. US 10,688,320 B2, IGRT uses images acquired prior to the treatment to guide the application of therapeutic irradiation during the treatment. The concentration of the imaging agent or therapeutic agent in the radiocarrier should be sufficient to provide satisfactory imaging. For example, when using an aqueous solution, the dose is about 1.0 to 100 millicuries. Imaging can be performed to provide a reference for guiding the target area to receive a calculated radiation flux from the therapeutic irradiation source. Similar uses of the compound as a reference can be used to guide surgical applications.

[0088] The amount of the compound of the present invention administered to a patient, or a formulation comprising a metal and a compound or a pharmaceutically acceptable salt thereof, depends on several physiological factors routinely used by the physician, including the nature of the procedure to be performed, the volume of the target tissue to be used for imaging or treatment, and the patient’s weight and medical history to be used to image or treat the patient with these compounds.

[0089] The embodiments provided herein are intended to illustrate the advantages of the present technology and to further assist those skilled in the art in preparing or using compounds or their salts, pharmaceutical compositions, derivatives, prodrugs, or tautomeric forms of the present technology. The embodiments herein are also intended to more fully illustrate preferred aspects of the present technology. These embodiments should in no way be construed as limiting the scope of the present technology as defined by the appended claims. These embodiments may include or combine any variations, aspects, or implementations of the present technology described above. The variations, aspects, or implementations described above may further each include or combine any or all other variations, aspects, or implementations of the present technology.

[0090] example Example 1 –Synthetic compounds Preparation of Int-1: Program 1: Synthesis of tert-butyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (2): Dioxane (4 M, 20 mL) containing HCl was added dropwise to a solution of 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoate tert-butyl ester (2.0 g, 3.81 mmol) in DCM (10 mL) at 0 °C, and the reaction mixture was stirred at 0 °C for 2 h. The solvent was evaporated, and the residue was co-evaporated with hexane and dried under vacuum to give an HCl salt of the product as a white solid (1.62 g, 100%). The crude product was used in the next reaction without further purification. LCMS:C 25 H 32 N₂O₄: m / z: 424.53, measured value m / z = 425.3 [M+H] + .

[0091] Procedure 2: Synthesis of tert-butyl hexanoate (4): 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)-butyrylamino)hexanoate In an ice bath, DIC (0.71 ml, 4.47 mmol) was added dropwise to a solution of 4-(4-isobutylphenyl)butyric acid (1.0 g, 4.57 mmol) in 10 ml of cooled DCM. The mixture was stirred for 30 min and filtered. The filtrate was added to a solution of tert-butyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (1.62 g, 3.81 mmol) in 5 ml of DCM, followed by the addition of additional DIC (0.66 ml, 3.81 mmol). The mixture was stirred again for 30 min. The solvent was evaporated under reduced pressure, and the crude product was purified by rapid chromatography (EtOAc / hexane, eluted in 50% EtOAc / hexane) to give a product as a white solid (1.93 g, 82% yield). LCMS: C 39 H 50 N₂O₅: m / z: 626.82, measured value m / z = 627.4 [M+H] + .

[0092] Procedure 3: Synthesis of tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butyrylamino)hexanoate (5): Diethylamine (8 ml) was added to a solution of tert-butyl hexanoate (0.95 g, 1.51 mmol) in THF (8 ml) at room temperature. The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated, the residue was washed with hexane (2x) and dried under vacuum to give the product (613 mg, 100%). This compound was used as is in the following chemical transformations. LCMS: C 24 H 40 N₂O₃: m / z: 404.59, measured value m / z = 405.4 [M+H] + .

[0093] Procedure 4: Synthesis of tert-butyl 33-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-3,31-dioxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonoxy-4,32-diazatriatetradecane-34-oic acid (7): To a solution of 3-oxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonoxy-4-azatriacontane-31-acid (872 mg, 1.51 mmol) in 10 mL of DCM, EDCI·HCl (434 mg, 2.27 mmol) and HOBt (203 mg, 1.51 mmol) were added, and the reaction mixture was stirred at room temperature for 5 min. DCM (10 mL) containing tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butyrylamino)hexanoate (613 mg, 1.51 mmol) and DIEA (0.8 mL, 4.53 mmol) were added sequentially, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with 20 mL of DCM and then washed with water. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by rapid chromatography (MeOH / DCM, eluting in DCM containing 3-4% MeOH) to give the product (1.0 g, yield 69%), as a colorless liquid. LCMS: C 51 H 83 N3O 14 m / z: 962.22, measured value m / z = 962.9 [M+H] + .

[0094] Procedure 5: Synthesis of tert-butyl 1-amino-29-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontane-30-olate (8): Palladium / carbon (10%, 0.2 g, 67% RH) was suspended in a solution of 33-(4-(4-(4-isobutyl-phenyl)butyrylamino)butyl)-3,31-dioxo-1-phenyl-2,7,10,13,16,19,22,25,28-nonoxy-4,32-diazatriacontane-34-oic acid tert-butyl ester (1.0 g, 1.03 mmol) in MeOH (25 ml). The suspension was stirred for 2 h at room temperature under H2 and balloon pressure. The mixture was filtered through diatomaceous earth, and the filtrate was evaporated to give a product as a white solid (850 mg, 100%). The crude product was used for the next step without further purification. LCMS: C 43 H 77 N3O 12 m / z: 828.08, measured value m / z = 828.7 [M+H] + .

[0095] Procedure 6: Synthesis of 10-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-41-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonoxy-5,12,40-triazatetane-42-acid tert-butyl ester (10): Add EDCI.HCl (0.29 g, 1.53 mmol) and HOBt (0.14 g, 1.02 mmol) to a solution of 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid (0.48 g, 1.02 mmol) in 10 mL of DCM, and stir the reaction mixture at room temperature for 5 min. Then, add DCM (10 mL) containing tert-butyl 1-amino-29-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-27-oxo-3,6,9,12,15,18,21,24-octaoxa-28-azatriacontane-30-oic acid (0.85 g, 1.02 mmol) and DIEA (0.53 mL, 3.07 mmol), and stir the mixture at room temperature for 16 hours. The reaction mixture was diluted with 20 ml of DCM and washed with water. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by rapid chromatography (MeOH / DCM, eluting in DCM containing 5% MeOH) to give a white solid (0.66 g, 50% yield). LCMS: C 69 H 107 N5O 17 m / z: 1278.61, measured value m / z = 1279.1 [M+H] + .

[0096] Procedure 7: Synthesis of 10-amino-41-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonoxy-5,12,40-triazatetrazo-dodecane-42-acid tert-butyl ester (11): Diethylamine (7 ml) was added to a solution of 0.66 g (0.51 mmol) of tert-butyl 10-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-41-(4-(4-(4-isobutylphenyl)-butyrylamino)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonoxy-5,12,40-triazatetrazododecane-42-oic acid tert-butyl ester (0.66 g, 0.51 mmol) in THF (7 ml). The reaction mixture was stirred at ambient temperature for 2 hours, and the solvent was evaporated. The residue was washed with hexane (2x) and dried under vacuum to give the product (700 mg, 100%). The compound was used in the next step without further purification. LCMS: C 54 H 97 N5O 15 m / z: 1056.37, measured value m / z = 1057.1 [M+H] + .

[0097] Procedure 8: Synthesis of 18-(4-((tert-butoxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)-butyrylamino)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecane-4,17,20,48-tetraazapentacontane-50-acid tert-butyl ester (13): Add EDCI.HCl (0.18 g, 0.93 mmol) and HOBt (0.08 g, 0.62 mmol) to a solution of 3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecane-16-acid (0.22 g, 0.62 mmol) in DCM (8 ml), and stir the reaction mixture at room temperature for 5 min. DCM (5 ml) and DIEA (0.32 ml, 1.87 mmol) containing 10-amino-41-(4-(4-(4-isobutylphenyl)-butyrylamino)butyl)-2,2-dimethyl-4,11,39-trioxo-3,15,18,21,24,27,30,33,36-nonoxy-5,12,40-triazatetrazo-dodecane-42-oic acid tert-butyl ester (0.66 g, 0.62 mmol) were added sequentially, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with 20 ml of DCM and washed with water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by rapid chromatography (MeOH / DCM, washed in DCM containing 8-10% MeOH) to give a product as a white solid (0.70 g, 50% yield). LCMS: C 71 H 120 N6O 21 m / z: 1393.74, measured value m / z = 1394.7 [M+H] + .

[0098] Procedure 9: Synthesis of 18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecane-4,17,20,48-tetraazapentacontane-50-acid tert-butyl ester (14): A solution of 18-(4-((tert-butoxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecane-4,17,20,48-tetraazapentacontan-50-oic acid tert-butyl ester (0.70 g, 1.22 mmol) in 90% formic acid aqueous solution (10 ml) was stirred at ambient temperature for 2 h. Complete conversion was detected by LCMS. The solvent was evaporated, and the residue was co-evaporated with ACN (3x) and toluene (3x). The residue was dissolved in a mixture of MeOH / water (1:1, 25 ml) and treated with AmberLite HPR550 ion exchange resin (OH- form) at ambient temperature for 15 min. The resin was filtered and washed with MeOH and water. The filtrate was evaporated to give the target material (free base, 0.62 g, 95% yield) as a colorless liquid. The crude amine was used in this step without further purification. LCMS: C 66 H 112 N6O 19 m / z: 1293.62, measured value m / z = 1294.1 [M+H] + .

[0099] Procedure 10: Synthesis of 2,2',2''-(10-(39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecaneamino)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctaoctachioalkyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester (15): DIEA (0.20 ml, 1.15 mmol) was added to a solution of 18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butyrylamino)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecane-4,17,20,48-tetraazapentacontane-50-oic acid tert-butyl ester (0.62 g, 0.48 mmol), DOTA-tris(tert-butyl ester) (0.27 g, 0.48 mmol), and PyBop (0.30 g, 0.56 mmol) in DMSO (8 ml), and the reaction mixture was stirred at ambient temperature for 16 h. Water (20 ml) was added, and the mixture was extracted with EtOAc (30 ml). The separated organic layer was washed with water (7x) and brine (8x) to remove excess PyBOP. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to give the target product (820 mg, 93% yield) as a colorless liquid. The compound was used in the next step without further purification. LCMS: C 94 H 162 N 10 O 26 m / z: 1848.34, measured value m / z = 1849.7 [M+H] + .

[0100] Procedure 11: Synthesis of 2,2',2''-(10-(8-(3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionamide)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctaoctadecanyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester (16): Palladium / carbon (10%, 0.12 g, 67% humidity) was suspended in a solution of 2,2',2''-(10-(39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecaneamino)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctaoctachioalkyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate tritert-butyl (500 mg, 0.270 mmol) in MeOH (15 ml). The suspension was stirred for 2 h at room temperature under H2 and balloon pressure. The mixture was filtered through diatomaceous earth, and the filtrate was evaporated to give the target compound (387 mg, 83% yield) as a colorless liquid. The crude product was used in the next step without further purification. LCMS: C 86 H 156 N 10 O 24 m / z: 1714.21, measured value m / z = 1714.7 [M+H] + .

[0101] Procedure 12: Synthesis of 2,2',2''-(10-(8-(3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionamide)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctaoctachioyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (17): TFA (2 ml) was added to a solution of 2,2',2''-(10-(8-(3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propionamide)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctaoctachioyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (387 mg, 0.225 mmol) in DCM (4 ml). The reaction mixture was stirred at 40 °C for 2 h and the solvent was evaporated under reduced pressure. The residue was washed with hexane (2x) and diethyl ether (2x) to give the product (340 mg, 100%) as a white powder. The crude product was used in the next step without further purification. LCMS: C 70 H124 N 10 O 24 m / z: 1489.79, measured value m / z = 1490.5 [M+H] + .

[0102] Preparation of Int 2: Program 13: Synthesis of tert-butyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (2): Dioxane (4 M, 20 mL) containing HCl was added dropwise to a solution of tert-butyl 2-(((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)-amino)hexanoate (2.0 g, 3.81 mmol) in DCM (10 mL) at 0 °C, and the reaction mixture was stirred at 0 °C for 2 h. The solvent was evaporated, and the residue was co-evaporated with hexane and dried under vacuum to give an HCl salt of the product as a white solid (1.62 g, 100%). The crude product was used for the next step without further purification. LCMS: C 25 H 32 N₂O₄: m / z: 424.53, measured value m / z = 425.3 [M+H] + .

[0103] Program 14: Synthesis of tert-butyl hexanoate (4): 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-(4-(4-isobutylphenyl)butamido)hexanoate DIC (0.71 ml, 4.47 mmol) was added dropwise to a solution of 4-(4-isobutylphenyl)butyric acid (1.0 g, 4.57 mmol) in 10 ml of cooled DCM in an ice bath. The mixture was stirred for 30 minutes and filtered. The filtrate was added to a solution of tert-butyl 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-aminohexanoate (1.62 g, 3.81 mmol) in 5 ml of DCM, and then DIEA (0.66 ml, 3.81 mmol) was added dropwise. The mixture was stirred for another 30 minutes. The solvent was evaporated under reduced pressure, and the crude product was purified by rapid column chromatography (EtOAc / hexane, eluted with 50% EtOAc / hexane) to give a white solid (1.93 g, 82% yield). LCMS: C 39 H 50N₂O₅: m / z: 626.82, measured value m / z = 627.4 [M+H] + .

[0104] Program 15: Synthesis of tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butamido)hexanoate (5): Diethylamine (8 mL) was added to a solution of tert-butyl hexanoate (0.95 g, 1.51 mmol) in THF (8 mL) at room temperature. The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated, and the residue was washed twice with hexane and dried under vacuum to give the product (613 mg, 100%). The compound was used directly for the next chemical transformation. LCMS: C 24 H 40 N₂O₃: m / z: 404.59, measured value m / z = 405.4 [M+H] + .

[0105] Procedure 16: Synthesis of tert-butyl hexanoate (7): 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexamido)-6-(4-(4-isobutylphenyl)butamido)tert-butyl hexanoate EDCI·HCl (0.92 g, 4.78 mmol) and HOBt (0.43 g, 3.19 mmol) were added to a solution of 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid (1.50 g, 3.19 mmol) in 15 mL of DCM, and the reaction mixture was stirred at room temperature for 5 min. Then, DCM (10 mL) containing tert-butyl 2-amino-6-(4-(4-isobutylphenyl)butamido)hexanoate (1.29 g, 3.19 mmol) and DIEA (1.66 mL, 9.57 mmol) were added sequentially, and the mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with DCM (20 mL) and washed with water. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by rapid column chromatography (EtOAc / hexane, eluted with 75% EtOAc / hexane) to obtain a glassy solid (1.18 g, 43% yield). LCMS: C 12 H 24 N₂O₃: m / z: 855.11, measured value m / z = 245.4 [M+H]+ .

[0106] Program 17: Synthesis of tert-butyl hexanoate 2-(2-amino-6-((tert-butoxycarbonyl)amino)hexamido)-6-(4-(4-isobutylphenyl)butamido)hexanoate (8): Diethylamine (10 mL) was added to a solution of tert-butyl hexanoate (1.18 g, 1.38 mmol) in THF (10 mL) at room temperature. The reaction mixture was stirred at ambient temperature for 2 hours. The solvent was evaporated, and the residue was washed with hexane (2x) and dried under vacuum to give the product (900 mg, 100%). The compound was used in the next reaction without further purification. LCMS: C 35 H 60 N4O6: m / z: 632.87, measured value m / z = 633.9 [M+H] + .

[0107] Procedure 18: Synthesis of 18-(4-((tert-butyloxycarbonyl)amino)butyl)-21-(4-(4-(4-isobutylphenyl)butamido)butyl)-3,16,19-trioxo-1-phenyl-2,7,10,13-tetraoxa-4,17,20-triazadocosane-22-acid tert-butyl ester (10): EDCI·HCl (0.41 g, 2.13 mmol) and HOBt (0.19 g, 1.42 mmol) were added to a solution of 3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecane-16-acid (0.50 g, 1.42 mmol) in 8 mL of DCM, and the reaction mixture was stirred at room temperature for 5 min. Then, 10 mL of DCM containing 2-(2-amino-6-((tert-butoxycarbonyl)amino)hexamido)-6-(4-(4-isobutylphenyl)butamido)hexanoate tert-butyl ester (0.90 g, 1.42 mmol) and DIEA (0.72 mL, 4.27 mmol) were added sequentially, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with 20 mL of DCM and washed with water. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by rapid column chromatography (MeOH / DCM, eluting with DCM containing 3-4% MeOH) to obtain a colorless oil (0.95 g, 69% yield). LCMS: C52 H 83 N5O 12 m / z: 970.24, measured value m / z = 970.8 [M+H] + .

[0108] Program 19: Synthesis of tert-butyl 1-amino-14-(4-((tert-butyloxycarbonyl)amino)butyl)-17-(4-(4-(4-isobutylphenyl)butamido)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-olate (11): Palladium / carbon (10%, 0.040 g, wet, 67%) was suspended in a solution of 18-(4-((tert-butoxycarbonyl)amino)butyl)-21-(4-(4-(4-isobutylphenyl)butamido)butyl)-3,16,19-trioxo-1-phenyl-2,7,10,13-tetraoxa-4,17,20-triazadocosahexadecanoic-22-oic acid tert-butyl ester (160 mg, 0.165 mmol) in MeOH (5 ml). The suspension was stirred under balloon pressure at room temperature in H2 for 2 h. The mixture was then filtered through diatomaceous earth, and the filtrate was evaporated to give the target compound (150 mg, 100%) as a white solid. The crude product was used in the next step without further purification. LCMS: C 44 H 77 N5O 10 m / z: 836.11, measured value m / z = 836.80 [M+H] + . Procedure 20: Synthesis of tert-butyl 18-(4-((tert-butyloxycarbonyl)amino)butyl)-1-(9H-fluorene-9-yl)-21-(4-(4-(4-isobutylphenyl)butamido)butyl)-3,16,19-trioxo-2,7,10,13-tetraoxa-4,17,20-triazadocosane-22-oic acid (12): To a solution of 1-amino-14-(4-((tert-butyloxycarbonyl)amino)butyl)-17-(4-(4-(4-isobutylphenyl)butamido)butyl)-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-oic acid tert-butyl ester (150 mg, 0.179 mmol) in DCM (4 ml) at room temperature, FmocOSu (73 mg, 0.215 mmol) and DIPEA (0.1 ml, 0.538 mmol) were added, and the reaction mixture was stirred at room temperature for 16 h. Water was added, and the mixture was extracted with DCM. The organic layer was dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The crude product was purified by rapid column chromatography (EtOAc / hexane, eluted with hexane containing 50% EtOAc) to give a white solid (145 mg, 77% yield). LCMS: C 59 H 87 N5O 12 m / z: 1058.35, measured value m / z = 1059.0 [M+H] + .

[0109] Procedure 21: Synthesis of 18-(4-aminobutyl)-1-(9H-fluorene-9-yl)-21-(4-(4-(4-isobutylphenyl)butamido)butyl)-3,16,19-trioxo-2,7,10,13-tetraoxa-4,17,20-triazadocosane-22-acid (13): TFA (1 ml) was added to a solution of 18-(4-((tert-butyloxycarbonyl)amino)butyl)-1-(9H-fluorene-9-yl)-21-(4-(4-(4-isobutylphenyl)butamido)butyl)-3,16,19-trioxo-2,7,10,13-tetraoxa-4,17,20-triazadocosahexadecane-22-oic acid tert-butyl ester (145 mg, 0.137 mmol) in DCM (3 ml) at 0 °C, and the reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure, and the crude product was purified by HPLC. The desired fractions were combined and lyophilized to give the target product (65 mg, 53%) as a grayish-white solid. LCMS: C 50 H 71 N5O 10 m / z: 902.13, measured value m / z = 902.9 [M+H] + Program 22: Synthesis of 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-(4-isothiocyanate phenethoxy)pyridinecarboxylic acid (16): Na₂CO₃ (44 mg, 0.42 mmol) was added to a solution of 4-(4-aminophenethoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)pyridinecarboxylic acid. 2×TFA (120 mg, 0.14 mmol) in DCM (3.5 mL). The reaction mixture was stirred at 40 °C for 15 min (until a homogeneous solution was obtained). The reaction mixture was cooled to room temperature, and 0.5 mL of DCM containing O,O-di(pyridin-2-yl)thiocarbonate (36 mg, 0.15 mmol) was added. After LCMS analysis showed that the amine had been consumed, the solids were removed by filtration, and volatiles were removed under reduced pressure to give product 16 (100 mg, 100%) as a yellow oil. LCMS:C 35 H 43 N5O9S: m / z: 709.81, measured value m / z = 710.4 [M+H] + .

[0110] Procedure 23: Synthesis of 4-(4-(3-(18-((1-carboxy-5-(4-(4-isobutylphenyl)butamido)pentyl)carbamoyl)-1-(9H-fluorene-9-yl)-3,16-dioxo-2,7,10,13-tetraoxa-4,17-diazacosano-22-yl)thiourea)phenethoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)pyridinecarboxylic acid (17): Add Na2CO3 (8 mg, 0.069 mmol) and 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-(4-isothiocyanate phenethoxy)pyridinecarboxylic acid (20 mg, 0.027 mmol, dissolved in 0.5 mL of DMSO:H2O) to a solution of 18-(4-aminobutyl)-1-(9H-fluorene-9-yl)-21-(4-(4-(4-isobutylphenyl)butamido)methyl)-3,16,19-trioxo-2,7,10,13-tetraoxa-4,17,20-triazacosano-22-oic acid (21 mg, 0.023 mmol) in DMSO:H2O (0.5 mL:0.5 mL) to a solution of 18-(4-aminobutyl)-1-(9H-fluorene-9-yl)-21-(4-(4-(4-isobutylphenyl)butamido)methyl)-3,16,19-trioxo-2,7,10,13-tetraoxa-4,17,20-triazacyclooctadecane-7-yl)methyl)-4-(4-isothiocyanate phenethoxy)pyridinecarboxylic acid (20 mg, 0.027 mmol, dissolved in 0.5 mL of DMSO:H2O) in DMSO:H2O (0.5 mL):H2O (0.5 mL). The mixture was added to DMSO and stirred at room temperature for 2 h. LCMS analysis showed complete amine consumption. The solid was removed by filtration, and the crude product was purified by HPLC. The desired fractions were combined and lyophilized to give a product (28 mg, 76%) as a grayish-white solid. LCMS:C 85 H 114 N 10 O 19 S: m / z: 1611.94, measured value m / z = 1612.4 [M+H] + .

[0111] Procedure 24: Synthesis of 4-(4-(3-(1-amino-14-((1-carboxy-5-(4-(4-isobutylphenyl)butamido)pentyl)carbamoyl)-12-oxo-3,6,9-trioxa-13-azaoctadecane-18-yl)thiourea)phenethoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)pyridinecarboxylic acid (18): Diethylamine (0.5 ml) was added to a solution of 4-(4-(3-(18-((1-carboxy-5-(4-(4-isobutylphenyl)butamido)pentyl)carbamoyl)-1-(9H-fluorene-9-yl)-3,16-dioxo-2,7,10,13-tetraoxa-4,17-diazacosano-22-yl)thioureo)phenethoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)pyridinecarboxylic acid (28 mg, 0.017 mmol) in THF (0.5 ml) at room temperature. The reaction mixture was stirred at room temperature for 2 hours, the solvent was evaporated, and the crude residue was purified by HPLC. The desired fractions were combined and lyophilized to give a product (13 mg, 54%) as a grayish-white solid. LCMS: C 70 H 104 N 10 O 17 S: m / z: 1389.70, measured value m / z = 1390.2 [M+H] + .

[0112] Synthetic Int-3 Procedure 25: Preparation of tert-butyl (S)-2-[(S)-2-(3-{2-[2-(2-(2-aminoethoxy)ethoxy]ethoxy}propionylamino)-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoic acid: Fmoc-L-Lys (Boc)-OtBu, Fmoc-L-Lys(Boc)-OH (47 g, 100 mmol) was dissolved in DCM (470 ml), and diisopropylethylamine (25 ml) was added at -30 °C. The reaction mixture was stirred at -30 °C for 5 min, and then Boc anhydride (30.1 g, 140 mmol) was slowly added. The reaction mixture was stirred at -30 °C for another 30 min, followed by the addition of N,N-dimethylaminopyridine (1.7 g, 14 mmol) at -30 °C. The reaction mixture was slowly heated to 0 °C over 2 hours and stirred at 0 °C for another 2 hours. At this point, LCMS analysis showed that the initial carboxylic acid in the reaction mixture had been completely consumed. The reaction mixture was cooled to -30 °C and 1 M HCl aqueous solution was slowly added to obtain pH ~3 (the temperature of the mixture was maintained below -20 °C). The resulting two-phase mixture was separated, and the organic layer was washed successively with water (200 ml) and 0.1 M NaHCO3 (200 ml), dried over MgSO4, filtered, and evaporated to dryness. The residue was diluted with 30 mL of ethyl acetate, heated to reflux, and then diluted with 300 mL of hexane. The mixture was stirred at room temperature for 2 hours and filtered. The precipitate was washed with 100 mL of 20% ethyl acetate / hexane and kept under vacuum overnight to remove residual solvent. Yield = 23 g (44%).

[0113] Program 26: Fmoc-L-Lys (Boc)-OtBu Dioxane (275 ml) containing 4 M HCl was added to a solution of Fmoc-L-Lys(Boc)-OtBu (23 g, 44 mmol) in 130 ml DCM at -5 °C. The mixture was stirred at -5 °C to 0 °C for 2.5 h (until LCMS showed no starting material). The solvent was evaporated under vacuum without heating, then re-evaporated with 150 ml DCM, and the residual solvent was removed under vacuum at room temperature overnight. The product (22.6 g, 110%) was given as a white amorphous solid.

[0114] Program 27: (S)-2-[(9H-fluorene-9-yl)methoxycarbonylamino]-6-(hexanoylamino)tert-butyl hexanoate Diisopropylcarbodiimide (DIC, 16.7 g, 132 mmol) was added dropwise to a solution of hexanoic acid (30.7 g, 264 mmol) in DCM (120 ml) at 0 °C, and the reaction mixture was stirred at 0 °C for 30 min. The precipitated solid was removed by filtration. The filtrate was added to a suspension of Fmoc-L-Lys-OtBu hydrochloride (20.3 g, 44 mmol) in DCM (30 ml), followed by the addition of DIPEA (7.7 mL, 1 equivalent); the temperature was maintained between 0 °C and 5 °C during the addition. The reaction mixture was stirred at 5 °C for 30 min, at which point LCMS analysis showed no remaining starting material. The mixture was diluted with 150 ml of DCM, and the organic layer was washed with 0.5 M HCl solution (100 ml), washed with 0.1 M NaHCO3 solution (100 ml), dried over MgSO4, and evaporated. The residue was purified by rapid column chromatography (330 g silica gel column, hexane containing 0% to 50% ethyl acetate) to give the target compound (16.8 g, 73%) as a white glassy substance.

[0115] Program 28: (S)-2-amino-6-(hexanoylamino)hexanoate tert-butyl ester Diethylamine (92 ml, 900 mmol) was added to a solution of (S)-2-[(9H-fluorene-9-yl)methoxycarbonylamino]-6-(hexanoylamino)hexanoate tert-butyl ester (16 g, 30 mmol) in THF (200 ml) at 10 °C. The reaction mixture was stirred for 1 hour and evaporated to dryness under vacuum without heating. The residue was re-evaporated with toluene (100 ml) at 25–30 °C and purified by silica gel column chromatography (220 g column, DCM containing 0% to 15% MeOH) to give a viscous yellow oily product (7.1 g, 79% yield).

[0116] Program 29: (S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)tert-butyl hexanoate HOBt (3.45 g, 25.5 mmol) was added to a solution of CBZ-Lys(Boc)-OH (9.7 g, 25.5 mmol) in anhydrous DCM (100 ml), and the mixture was stirred at ambient temperature for 10 min. DCM containing (S)-2-amino-6-(hexanoylamino)hexanoate tert-butyl ester (6.9 g, 23 mmol), DIPEA (12.2 ml), and EDC (5.35 g) were added sequentially. The reaction mixture was stirred at ambient temperature for 4 hours (until LCMS showed that (S)-2-amino-6-(hexanoylamino)hexanoate tert-butyl ester was consumed), diluted with DCM (200 ml), washed with water (150 ml) and saturated NaCl solution (100 ml), and dried over anhydrous MgSO4. The organic solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (220 g column, hexane containing 15% to 80% ethyl acetate) to give a product (11.4 g, 75%) as an amorphous yellow solid.

[0117] Program 30: (S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)tert-butyl hexanoate A solution of (S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate tert-butyl ester (11 g, 16.6 mmol) in methanol (400 ml) was added with 10% palladium (50% wet weight) / activated carbon (2 g). The suspension was subjected to catalytic hydrogenation at ambient temperature (H2, 1 atm, overnight) (LCMS showed complete consumption of the feed after 12 h). The reaction mixture was filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure, evaporated twice with 200 ml of acetonitrile, and dried under high vacuum for 4 h to give a colorless, glassy product (8.96 g, 100%).

[0118] Program 31: (S)-2-[(S)-2-[3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionylamino]-6-(tert-Butyloxycarbonylamino)hexanoylamino]-6-(hexanoylamino)tert-butyl hexanoate HOBt (2.53 g, 18.7 mmol) was added to a solution of 3-(2-{2-[2-(benzyloxycarbonylamino)ethoxy]ethoxy}ethoxy)propionic acid (5.8 g, 16.6 mmol) in anhydrous DCM (50 mL), and the mixture was stirred at ambient temperature for 10 min. Subsequently, a solution of (S)-2-[(S)-2-amino-6-(tert-butoxycarbonylamino)hexanoylamino]-6-(hexanoylamino)hexanoate tert-butyl ester (8.9 g, 16.6 mmol) in DCM (50 mL), EDC (5.35 g), and DIPEA (12.2 mL) were added. The reaction mixture was stirred for 4 h (LCMS monitoring), diluted with DCM (200 mL), washed with water (150 mL) and saturated NaCl solution (100 mL), and dried over MgSO4. The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (220 g column, DCM containing 0% to 10% MeOH) to obtain the target product (9.63 g, 67%), which was a colorless glassy substance.

[0119] Procedure 32: Synthesis of 18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butamido)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecoxa-4,17,20,48-tetraazapentadecane-50-acid tert-butyl ester A solution of 0.70 g (1.22 mmol) of tert-butyl 18-(4-((tert-butyloxycarbonyl)amino)butyl)-49-(4-(4-(4-isobutylphenyl)butamido)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecanooxa-4,17,20,48-tetraazapentacontane-50-oic acid in 90% aqueous formic acid solution (10 ml) was stirred at ambient temperature for 2 h. The reaction transformation was detected by LCMS. The solvent was evaporated, and the residue was co-evaporated with ACN (3 times) and toluene (3 times). The residue was dissolved in a methanol / water mixture (1:1, 25 ml) and treated with AmberLite HPR550 (OH type) ion exchange resin at ambient temperature for 15 min. The resin was filtered and washed with MeOH and water. The filtrate was evaporated to give the target product (free base, 0.62 g, 95% yield) as a colorless liquid. The crude amine was used in subsequent steps without further purification. LCMS: C 66 H 112 N6O 19m / z: 1293.62, measured value m / z = 1294.1 [M+H] + .

[0120] Procedure 33: Synthesis of 2,2',2''-(10-(39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecanoamide)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraaza-tetraoctadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester (15): DIEA (0.20 ml, 1.15 mmol) was added to a solution of 18-(4-aminobutyl)-49-(4-(4-(4-isobutylphenyl)butamido)butyl)-3,16,19,47-tetraoxo-1-phenyl-2,7,10,13,23,26,29,32,35,38,41,44-dodecano-4,17,20,48-tetraazapentadecane-50-oic acid tert-butyl ester (0.62 g, 0.48 mmol), DOTA-tris(tert-butyl ester) (0.27 g, 0.48 mmol), and PyBop (0.30 g, 0.56 mmol) in DMSO (8 ml), and the reaction mixture was stirred at ambient temperature for 16 h. Water (20 ml) was added, and the mixture was extracted with EtOAc (30 ml). The separated organic layer was washed with water (7x) and brine (8x) to remove excess PyBOP. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to give the target product (820 mg, 93% yield) as a colorless liquid. This compound was used directly in the next step without further purification. LCMS: C 94 H 162 N 10 O 26 m / z: 1848.34, measured value m / z = 1849.7 [M+H] + .

[0121] Procedure 34: Synthesis of 2,2',2''-(10-(8-(3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propamido)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxo-3,10,38,44-tetraaza-tetraoctadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester (16): Palladium / carbon (10%, 0.12 g, wet, 67%) was suspended in a solution of 2,2',2''-(10-(39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azahexadecanoamido)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazatoctadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate tritert-butyl (500 mg, 0.270 mmol) in MeOH (15 ml). The suspension was stirred under balloon pressure at room temperature for 2 h in H2. The mixture was filtered through diatomaceous earth, and the filtrate was evaporated to give the target compound (387 mg, 83% yield) as a colorless liquid. The crude product was used in the next step without further purification. LCMS: C 86 H 156 N 10 O 24 m / z: 1714.21, measured value m / z = 1714.7 [M+H] + .

[0122] Procedure 35: Synthesis of 2,2',2''-(10-(8-(3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propamido)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxo-3,10,38,44-tetraaza-tetraoctadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (17): TFA (2 ml) was added to a solution of 2,2',2''-(10-(8-(3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propamido)-39-(tert-butoxycarbonyl)-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxo-3,10,38,44-tetraazaoctanoate tetradecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate tritert-butyl (387 mg, 0.225 mmol) in DCM (4 ml) at 0 °C. The reaction mixture was stirred at 40 °C for 2 h, and the solvent was evaporated under reduced pressure. The residue was washed with hexane (2x) and diethyl ether (2x) to give a white powdery product (340 mg, 100%). The crude product was used in the next step without further purification. LCMS:C 70 H 124 N 10 O 24 m / z: 1489.79, measured value m / z = 1490.5 [M+H] + .

[0123] Synthetic RTX-1392R RTX-1392R was prepared using Int-1 as the starting material and following the procedure: Procedure 36: Synthesis of 2,2',2''-(10-(39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(2-oxo-1-((4-(((R)-1-oxo-1-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methylenebenzo[d][1,3,2]dioxoboranecyclopentane) (19) pyrrolidine-1-yl prop-2-yl carbamoyl quinolin-7-yl oxy)-6,9,12-trioxa-3-azapentadecan-do)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctane-tetraalkyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid: Add 0.19 ml of DIPEA to a solution of 2,2',2''-(10-(8-(3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propamido)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxo-3,10,38,44-tetraaza-tetraoctadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (200 mg, 0.134 mmol) in DMF (2 ml) at 0 °C, and stir the reaction mixture at 0 °C for 5 min. DMF (1 ml) containing 2-((4-(((2R)-1-oxo-1-((2R)-2-((3aS,4R,6R)-3a,5,5-trimethylhexahydro-4,6-methylene-benzo[d][1,3,2]dioxoboronide-2-yl)pyrrolidine-1-yl)prop-2-yl)carbamoyl)quinolin-7-yl)oxy)acetic acid 2,3,5,6-tetrafluorophenyl ester (121 mg, 0.174 mmol) was added, and the reaction mixture was stirred at 0 °C for 0.5 h. After LCMS showed complete consumption of the amine, the crude product was purified by HPLC. The desired fractions were combined and lyophilized to give a product as a grayish-white solid (72 mg, 26%). LCMS: C 99 H 158 BN 13 O 30 m / z: 2021.19, measured value m / z = 1011.7 [M / 2+H] + .

[0124] Procedure 37: Synthesis of 2,2',2''-(10-(8-(1-((4-(((R)-1-((R)-2-boronpyrrolidine-1-yl)-1-oxopropyl-2-yl)carbamoyl)quinoline-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecanamido)-39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxa-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazatoctadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (RTX-1392R (20)): To 2,2',2''-(10-(39-carboxy-48-(4-isobutylphenyl)-2,9,37,45-tetraoxo-8-(2-oxo-1-((4-(((R)-1-oxo-1-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methylenebenzo[d][1,3,2]dioxoborherocyclopentene- MeB(OH)₂ (10 mg, 0.173 mmol) was added to a solution of 2-yl)pyrrolidine-1-yl)propyl-2-yl)carbamoyl)quinoline-7-yl)oxy)-6,9,12-trioxa-3-azapentadecanamido)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazatetrazoyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (70 mg, 0.034 mmol) in acetone (1.5 ml) and 0.2N HCl (1.5 ml) at room temperature, and the reaction mixture was stirred at room temperature for 30 min. After LCMS showed complete consumption of the starting material, the crude product was purified by HPLC. The desired fractions were combined and lyophilized to give a product (31 mg, 48%) as a grayish-white solid. LCMS:C 89 H 144 BN 13 O 30 m / z: 1886.98, measured value m / z = 943.9 [M-OH / 2] + .

[0125] Synthesize RTX-1401R: RTX-1401R was prepared using Int-2 as the starting material and following the procedure: Program 38: Synthesis of (R)-2-[(7-hydroxy-4-quinolinyl)carbonylamino]tert-butyl propionate DIPEA (4.6 ml, 2.5 equivalents) was added to 40 mL of DMF containing 7-hydroxy-4-quinolinecarboxylic acid (2.0 g, 10.5 mmol, 1 equivalent), (R)-Ala-tert-butyl hydrochloride (2.0 g, 11.5 mmol, 1.1 equivalents), and HATU (5.98 g, 15.75 mmol, 1.5 equivalents) at 5 °C, and the mixture was stirred at room temperature for 2 h. After the reaction was complete (indicated by LCMS), H2O was added, and the product was extracted with n-butanol. The organic layer was separated, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The residue was purified by rapid column chromatography (DCM containing 0-10% MeOH) to give compound 3 (2.01 g, 61% yield) as a grayish-white powder. ESI-MS calculated m / z 316.351, experimental 317.0 [M+H] + .

[0126] Program 39: Synthesis of (R)-2-({7-[(benzyloxycarbonyl)methoxy]-4-quinolinyl}carbonylamino)tert-butyl propionate Benzyl bromoacetate (4.4 g, 19.2 mmol, 3 equivalents) was added to a suspension of (R)-2-[(7-hydroxy-4-quinolinyl)carbonylamino]propionate tert-butyl ester (2.0 g, 6.4 mmol, 1 equivalent) and potassium carbonate (2.7 g, 19.2 mmol, 3 equivalents) in DMF at room temperature, and the mixture was stirred at 60 °C for 16 h. After completion, H2O was added and the product was extracted with EtOAc. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The residue was purified by rapid column chromatography (hexane containing 0-80% ethyl acetate) to give compound 5 (2 g, 67% yield) as a grayish-white powder. ESI-MS calculated m / z 464.509, experimental 465.1 [M+H] + .

[0127] Procedure 40: Synthesis of (R)-2-({7-[(benzyloxycarbonyl)methoxy]-4-quinolinyl}carbonylamino)propionic acid 20 ml of TFA / DCM (DCM:TFA, 1:2) was added to DCM containing (R)-2-({7-[(benzyloxycarbonyl)methoxy]-4-quinolinyl}carbonylamino)propionate (2.0 g, 4.3 mmol) at room temperature, and the mixture was stirred for 4 h. After completion, the solvent was evaporated under reduced pressure to give compound 6 (1.75 g, 100%) as a grayish-white solid, which could be used in the next step without further purification. ESI-MS calculated m / z value was 408.403, experimental value was 409.3 (M+1)+.

[0128] Procedure 41: Synthesis of (4-{[(R)-2-(2-{(1R,2S,8S)-2,9,9-trimethyl-3,5-dioxa-4-borazaricyclo[6.1.1.02,6]dec-4-yl}-1-pyrrolidinyl)-1-methyl-2-oxoethylamino]carbonyl}-7-quinolinoxy)benzyl acetate DIPEA (1.9 ml, 10.75 mmol, 2.5 equivalents) was added to a solution of (R)-2-({7-[(benzyloxycarbonyl)methoxy]-4-quinolinyl}carbonylamino)propionic acid (1.75 g, 4.3 mmol, 1 equivalent), (R)-boronyl-(+)-pinenediol hydrochloride (1.35 g, 4.73 mmol, 1.1 equivalents), and HATU (2.45 g, 6.45 mmol, 1.5 equivalents) in DMF (15 ml) at 5 °C. The mixture was stirred at room temperature for 2 h, at which point the reaction was indicated to be complete by LCMS. H2O was added and the product was extracted with EtOAc. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The residue was purified by rapid column chromatography (DCM containing 0-10% MeOH) to give compound 8 (1.75 g, 64% yield) as a grayish-white powder. ESI-MS calculated m / z value: 639.547; experimental value: 640.2 [M+H] + .

[0129] Procedure 42: Synthesis of (4-{[(R)-2-(2-{(1R,2S,8S)-2,9,9-trimethyl-3,5-dioxa-4-borazaricyclo[6.1.1.02,6]dec-4-yl}-1-pyrrolidinyl)-1-methyl-2-oxoethylamino]carbonyl}-7-quinolinoxy)acetic acid Activated palladium / carbon (5%, 0.4 g) was suspended in a solution of compound 8 (1.75 g, 2.75 mmol) in MeOH (150 mL). The suspension was stirred for 1 h at room temperature under a H2 atmosphere (40 psi). The mixture was then filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give 1.45 g of crude product. This substance was purified by reversed-phase HPLC (30-100% gradient, 0.1% formic acid / ACN) to give compound 9 (200 mg, 14% yield) as a grayish-white solid. ESI-MS calculated m / z 549.425, experimental 550.3 [M+H] + .

[0130] Program 43: Synthesis of 2-((4-(((2R)-1-(2-boropyrrolidone-1-yl)-1-oxopropyl-2-yl)carbamoyl)quinoline-7-yl)oxy)acetic acid (20): MeB(OH)₂ (10 mg, 0.173 mmol) was added to a solution of 2-((4-((((R)-1-oxo-1-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methylene-benzo[d][1,3,2]dioxoboronyl-2-yl)pyrrolidine-1-yl)prop-2-yl)carbamoyl)quinoline-7-yl)oxy)acetic acid (5 mg, 0.009 mmol) in a mixture of acetone (1.5 ml) and 0.2 N HCl (1.5 ml) at room temperature, and the reaction mixture was stirred at ambient temperature for 30 min. Water was added and the reaction mixture was extracted with DCM. The organic layer was separated, dried over Na₂SO₄, and concentrated under reduced pressure to give a colorless oily product (8 mg, 100%). The crude product was used in the next step without further purification. LCMS: C 19 H 22 BN3O7: m / z: 415.20, measured value m / z = 416.2 [M+H] + .

[0131] Program 44: Synthesis of ((R)-1-((R)-2-(7-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethoxy)quinoline-4-carboxamide-do)propionyl)pyrrolidine-2-yl)boronic acid (21): EDC·HCl (5.5 mg, 0.028 mmol) was added to a solution of 2-((4-((((2R)-1-(2-boropyrrolid-1-yl)-1-oxopropyl-2-yl)carbamoyl)quinolin-7-yl)-oxy)acetic acid (8 mg, 0.019 mmol) and 2,3,5,6-tetrafluorophenol (5 mg, 0.028 mmol) in 0.5 mL of DCM, and the reaction mixture was stirred at room temperature for 0.5 h. Water was added to the reaction mixture, and the mixture was extracted with DCM. The organic layer was separated, dried over Na2SO4, and concentrated under reduced pressure to give an activated ester (10 mg, 100%) as a white powder. The crude product was used in the next step without further purification. LCMS:C 25 H 22 BF4N3O7: m / z: 563.26, measured value m / z = 563.9 [M+H] + .

[0132] Procedure 45: Synthesis of 4-(4-(3-(1-((4-((((R)-1-((R)-2-boronpyrrolidine-1-yl)-1-oxopropane-2-yl)carbamoyl)quinoline-7-yl)oxy)-17-((1-carboxy-5-(4-(4-isobutylphenyl)butamido)pentyl)carbamoyl)-2,15-dioxo-6,9,12-trioxa-3,16-diazacotetradecane-21-yl)thiourea)phenethoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)pyridinecarboxylic acid (RTX-1401R (22): Add 6 μL of DIPEA to a solution of 4-(4-(3-(1-amino-14-((1-carboxy-5-(4-(4-isobutylphenyl)butamido)pentyl)carbamoyl)-12-oxo-3,6,9-trioxa-13-azaoctadecane-18-yl)thiourea)phenethoxy)-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)pyridinecarboxylic acid (6 mg, 0.0043 mmol) in DMF (0.3 ml) at 0 °C, and stir the reaction mixture at 0 °C for 5 min. DMF (0.1 ml) containing ((R)-1-((R)-2-(7-(2-oxo-2-(2,3,5,6-tetrafluorophenoxy)ethoxy)quinoline-4-carboxamido)propionyl)pyrrolidine-2-yl)boronic acid (3 mg, 0.0051 mmol) was added, and the reaction mixture was stirred at 0 °C for 0.5 h. After LCMS analysis showed complete consumption of the amine, the crude product was purified by HPLC. The desired fractions were combined and lyophilized to give the target product (2.5 mg, 32%) as a grayish-white solid. LCMS: C 89 H 124 BN 13 O 23 S: m / z: 1786.89, measured value m / z = 1769.4 [M-OH] + .

[0133] Synthesize RTX-1407S: RTX-1407S was prepared using Int-3 as the starting material and following the procedure.

[0134] The preparation of Int-3 was modified as follows.

[0135] In procedure 24, 2-(4-isobutylphenyl)acetic acid is used instead of hexanoic acid.

[0136] Procedure 46: Preparation of tert-butyl [2-((S)-2-cyanopyrrolidone-1-yl)-2-oxoethyl]carbamate: DIPEA (3.9 mL, 22.5 mmol) and Boc-Gly-OSu (2.26 g, 8.3 mmol) were added to a solution of (S)-pyrrolidine-2-nitrile hydrochloride (1 g, 7.5 mmol) in DCM (35 mL). The reaction mixture was stirred at ambient temperature for 14 h. The solvent was evaporated and the residue was dissolved in DCM, washed with water, dried over anhydrous Mg2SO4, and filtered. The filtrate was concentrated to 20% of the initial volume and loaded into a CombiFlash™ apparatus for elution (gradient 0–10% MeOH / DCM). The desired fractions were combined and evaporated to give the target product (1.1 g, 58%) as an amorphous solid.

[0137] Procedure 47: Preparation of (S)-1-(2-amino-acetyl)-pyrrolidine-2-nitrile: p-Toluenesulfonic acid monohydrate (1.25 g, 6.6 mmol) was added to a stirred solution of (2-((S)-2-cyanopyrrolidone-1-yl)-2-oxoethyl)carbamate tert-butyl ester (1.1 g, 4.4 mmol) in acetonitrile (20 ml), and the mixture was stirred at ambient temperature for 14 h. The solvent was removed under reduced pressure to give the toluenesulfonate of the target product (1.8 g, yield exceeding the theoretical value). The purity of the compound was sufficient for further chemical transformation.

[0138] Procedure 48: Preparation of (S)-N-(2-(2-cyanopyrrolidone-1-yl)-2-oxoethyl)-7-hydroxyquinoline-4-carboxamide: A solution of 7-hydroxyquinoline-4-carboxylic acid (307 mg, 1.62 mmol), HOBt (221 mg, 1.62 mmol), and TBTU (521 mg, 1.62 mmol) in DMF (15 mL) was stirred at room temperature for 5 min. A solution of (S)-1-(2-aminoacetyl)-pyrrolidine-2-onitrile (1.5-toluenesulfonate) (1.78 mmol) and DIPEA (0.74 mL, 4.86 mmol) in DMF (5 mL) was added to the activated ester solution, and the resulting mixture was stirred at room temperature for 2 h while monitoring the reaction progress by LCMS. After complete conversion, the reaction mixture was concentrated under reduced pressure, dissolved in a small amount of dichloromethane, and filtered. The filtrate was evaporated, dissolved in DCM, loaded onto a CombiFlash™ silica gel column, and purified (gradient 0-20% MeOH / DCM) to give the target product (422 mg, 80% yield) as a grayish-white solid.

[0139] Procedure 49: Preparation of (S)-2-((4-((2-(2-cyanopyrrolidone-1-yl)-2-oxoethyl)carbamoyl)quinoline-7-yl)oxy)tert-butyl acetate: A flask containing a DMF solution (15 mL) of 7-hydroxyquinoline-4-carboxylic acid [2-((S)-2-cyanopyrrolidone-1-yl)-2-oxoethyl]-amide (407 mg, 1.25 mmol), 2-tert-butyl glycolate (249 mg, 1.88 mmol), and triphenylphosphine (395 mg, 1.51 mmol) was cooled in an ice-water bath. Diisopropyl azodicarbonate (300 μL, 1.51 mmol) was added dropwise to the cooled reaction mixture. The ice-water bath was removed, and the resulting solution was stirred at room temperature and monitored by LCMS. After completion, the solvent was removed under reduced pressure, and the residue was dissolved in DCM, loaded onto a CombiFlash™ silica column (gradient 0-10% MeOH / DCM), and purified to give the target product (404 mg, 61% yield) as a glassy solid.

[0140] Procedure 50: Preparation of {4-[2-((S)-2-cyanopyrrolidone-1-yl)-2-oxoethylcarbamoyl]-quinoline-7-oxy}-acetic acid: TFA (1.51 ml, 19.64 mmol) was added dropwise to a solution of {4-[2-((S)-2-cyanopyrrolidone-1-yl)-2-oxoethylcarbamoyl]-quinoline-7-oxy}-tert-butyl acetate (141 mg, 0.322 mmol) in DCM (1.5 ml) at 0 °C. The temperature of the stirred reaction mixture was raised to room temperature over 1 h, at which point complete conversion was detected by LCMS. The solvent was evaporated at room temperature, and the residue was co-evaporated with toluene (x3) at 40 °C. The crude intermediate 2 was of sufficient purity for subsequent conversions, but was prone to hydrolysis upon prolonged storage. Therefore, the compound was prepared just before use.

[0141] Procedure 51: Synthesis of 2-[2-(3-{2-[2-(2-{2-[4-({(R)-2-[(S)-2-cyano-1-pyrrolyl]-1-methyl-2-oxoethylamino}carbonyl)-7-quinolinoxy]acetamido}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetamido}hexanoylamino]-6-[2-(p-isobutylphenyl)acetamido]tert-butyl hexanoate (3) Add [4-({( R )-2-[( S [2-Cyano-1-pyrrolyl]-1-methyl-2-oxoethylamino]carbonyl]-7-quinolinoxy]acetic acid (18 mg, 0.046 mmol, 1 equivalent) and DIPEA (25 µL, 0.699 mmol, 3 equivalents), HOBt (6.8 mg, 0.050 mmol, 1.1 equivalents) and EDCI (7.8 mg, 0.050 mmol, 1.1 equivalents). The mixture was stirred at room temperature for 16 h and then diluted with DCM (5 ml) and water (5 ml). The aqueous layer was extracted with DCM (3 × 5 ml), and the combined organic layers were dried over Na2SO4 and concentrated. The residue was purified by rapid column chromatography (DCM containing 0–20% MeOH) to give the product (62 mg, 82%) as a colorless solid. LCMS: C 85 H 133 N 13 O 19 m / z: 1641.04, measured value m / z = 1642.32 [M+H] + .

[0142] Program 52: Synthesis of 2-[2-(3-{2-[2-(2-{2-[4-({(R)-2-[(S)-2-cyano-1-pyrrolyl]-1-methyl-2-oxoethylamino}carbonyl)-7-quinolinoxy]acetamido}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetamido}hexanoylamino]-6-[2-(p-isobutylphenyl)acetamido]hexanoic acid (RTX 1407S) TFA (283 µL, 3.70 mmol) was added to a solution of 2-[2-(3-{2-[2-(2-{2-[4-({(R)-2-[(S)-2-cyano-1-pyrrolyl]-1-methyl-2-oxoethylamino}carbonyl)-7-quinolinoxy]acetamido}ethoxy)ethoxy]ethoxy}propionylamino)-6-{2-[4,7,10-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraaza-1-cyclododecyl]acetamido}hexanoylamino]-6-[2-(p-isobutylphenyl)acetamido]tert-butyl hexanoate (62 mg, 0.037 mmol, 1 equivalent) in DCM (0.62 mL). The resulting mixture was stirred for 10 h. Volatile substances were removed and the residue was purified by HPLC to give a product as a colorless solid (35.8 mg, 68%). LCMS: C 69 H 101 N 13 O 19 m / z: 1416.62, measured value m / z = 1417.60 [M+H] + Program 53: Synthesis of N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-acetyllysine tert-butyl ester Acetic anhydride (524 μl, 5.51 mmol) was added to a stirred solution of 5-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-6-(tert-butoxy)-6-oxohexane-1-ammonium chloride (3 g, 4.6 mmol) and diisopropylethylamine (1.76 mL, 10.12 mmol) in DCM (24 mL). The mixture was stirred at room temperature for 90 min, at which point LCMS indicated conversion. The reaction mixture was diluted with EtOAc, washed with water and brine, dried over anhydrous MgSO4, and filtered. Volatile substances were removed by evaporation under reduced pressure to give the target compound (2.9 g, 91%) as a grayish-white solid.

[0143] Procedure 54: Synthesis of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonic acid-1-(3-(trimethylammonium)-propyl)-indoline-2-ethylene)-ethylidene)-2-(4-(3-((2,5-dioxopyrrolidone-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonium)propyl)-3H-indo-1-onium-5-sulfonate: Add 130 mg (0.3 mmol) of dipyrrolidinyl (N-succinimideoxy)carbophosphate to a solution of ZW800-1 (100 mg, 0.105 mmol) in anhydrous DMSO (10 mL), followed by dropwise addition of N,N-diisopropylethylamine (0.2 mL, 1.1 mmol) at room temperature. Stir the reaction mixture at ambient temperature for 16 hours, at which point complete conversion was detected by LCMS. Treat the reaction mixture with a 1:1:1 solution of ethanol:ethyl acetate:acetone (150 mL) and 0.1% trifluoroacetic acid (0.9 mL), mix, and allow to stand for 30 min. The solid was filtered and vacuum dried to obtain 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonic acid-1-(3-(trimethylammonium)-propyl)indoline-2-ethylene)ethylidene)-2-(4-(3-((2,5-dioxopyrrolidone-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonium)propyl)-3H-indo-1-onium-5-sulfonate (51 mg, 46%), which was used directly in the next step without further purification. LCMS: C 46 H 61 N9O 12 m / z: 1040.32, measured value m / z = 1040.5 [M] + .

[0144] Program 55: 2-((E)-2-((E)-3-(2-((E)-3,3-dimethyl-5-sulfonic acid-1-(3-(trimethylammonium)propyl)indoline-2-ethylene)ethylidene)-2-(4-(3-((2,5-dioxopyrrolidone-1-yl)oxy)-3-oxopropyl)phenoxy)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-(trimethylammonium)propyl)-3H-indo-1-onium-5-sulfonate (0.070 g, 0.067 mmol) was dissolved in DMSO (1 N6-acetyl-N2-((1-((4-((((R)-1-((S)-2-cyanopyrrolidone-1-yl)-1-oxopropyl-2-yl)carbamoyl)quinoline-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-acyl)-L-lysyl)-L-lysine (0.040 g, 0.045 mmol) was added to a solution of DMSO (1 mL) at room temperature. N,N-diisopropylethylamine (0.070 g, 0.067 mmol) was added, and the mixture was stirred for 16 h. Complete conversion was detected by LCMS. After the reaction was complete, volatile substances were removed, and the residue was purified by HPLC to obtain the target product.

[0145] Synthesize RTX-1371R: Following a procedure similar to that of RTX-1401R, Int-3 was used as the starting material for preparation. The preparation of Int-3 involved the following modifications: In procedure 27, 2-(4-isobutylphenyl)acetic acid is used instead of hexanoic acid.

[0146] Synthetic RTX-1384S It was prepared according to a procedure similar to that used for preparing Int-2, but with the following modifications: Use program 53 instead of program 14.

[0147] The product from procedure 19 was subjected to procedure 51 to attach a quinoline, and procedure 52 to remove the protecting group. This product was then used for the further synthesis of RTX-1384S. Subsequently, procedures 54 and 55 were performed to attach a fluorophore.

[0148] Synthesize RTX-1391R: Following a procedure similar to that of RTX-1401R, Int-3 was used as the starting material for preparation. The preparation of Int-3 involved the following modifications: In procedure 27, 2-(4-isobutylphenyl)butyric acid is used instead of hexanoic acid.

[0149] Synthetic RTX-1400R Following a procedure similar to that of RTX-1401R, Int-2 was used as the starting material for preparation. The preparation of Int-2 involved the following modifications: In procedure 14, 2-(4-isobutylphenyl)acetic acid is used instead of 2-(4-isobutylphenyl)butyric acid.

[0150] Synthetic RTX-1402R Following a procedure similar to RTX-1392, Int-1 was used as the starting material for preparation. The preparation of Int-1 involved the following modifications: Use procedure 23 instead of procedure 10 to install Macropa chelating agent.

[0151] Synthetic RTX-1411R Program 56: (N6-((benzyloxy)carbonyl)-N2-(2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29-nonoxa-5-azatridodecane-32-acyl)lysine tert-butyl ester) (3): DIPEA (2.2 equivalents) was added dropwise to a mixture of 1 (1 equivalent), 2 (1.1 equivalent), and HATU (1.2 equivalent) in DMF at 0 °C. The resulting reaction mixture was stirred at room temperature for 3 h. The completion of the reaction was monitored by LCMS. The solvent was evaporated to dryness, and the resulting crude reaction mixture was purified by Combi Flash using hexane containing 0–100% EtOAc (quantitative yield). LCMS: C 42 H 73 N3O 15 m / z: 859.50, measured value m / z = 860.6 [M+H] + .

[0152] Program 57: (2,2-Dimethyl-4-oxo-3,8,11,14,17,20,23,26,29-nonoxa-5-azatridodecane-32-acyl)lysine tert-butyl ester (4): 10% palladium / activated carbon (100 mg) was added to a solution of 0.5 g of 3 g in methanol (10 ml). The suspension was subjected to catalytic hydrogenation (H2, under balloon pressure) at ambient temperature for 3 h (LCMS control – consumption of starting material). The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The residue was dried under high vacuum to give the target product as a colorless liquid (quantitative). LCMS: C 34 H 67 N3O 13 m / z: 825.92, measured value m / z = 826.9 [M+H] + .

[0153] Program 58: 2,5-dioxopyrrolidine-1-yl ester of 4-(4-iodophenyl)butyric acid (5): N-hydroxysuccinimide (1.3 equivalents) was added to a stirred solution of iodophenylbutyric acid (2.0 g, 1 equivalent) in DCM (20 mL) at 0 °C. The reaction mixture was stirred at the same temperature for 5 min, and EDC·HCl (1.3 equivalents) was added. The resulting solution was stirred at room temperature for 1 h (the reaction progress was monitored by TLC). The reaction mixture was diluted with DCM (20 mL), washed with water (50 mL), separated, and the organic layer was dried over Na₂SO₄ and evaporated. The residue was washed with diethyl ether (2 × 20 mL) to give compound 5 (2.0 g) as a grayish-white solid.

[0154] Program 59: N2-(2,2-dimethyl-4-oxo-3,8,11,14,17,20,23,26,29-nonoxa-5-azatridodecane-32-acyl)-N6-(4-(4-iodophenyl)butyryl)lysine tert-butyl ester (6): 2,5-dioxopyrrolidine-1-yl ester of 4-(4-iodophenyl)butyric acid (5) (1.0 equivalent) was added to a solution of 4 (2.0 g, 1 equivalent) in DCM (25 ml) at room temperature, followed by the addition of DIPEA (1.2 equivalent). The reaction mixture was stirred for 30 minutes. After the reaction was complete (the reaction progress was monitored by LCMS), the solvent was evaporated under reduced pressure, and the crude residue was purified by Combi Flash using MeOH:DCM (0–20%) to give compound 6 (1.8 g), which was a light brown viscous oil. LCMS:C 44 H 76 IN3O 14m / z: 997.44, measured value (loss of tert-butyl) m / z = 471.45 [M / 2+H] + .

[0155] Program 60: N2-(1-amino-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-acyl)-N6-(4-(4-iodophenyl)butyryl)lysine(7): TFA (10 ml) was added to a stirred solution of 6 (1.8 g, 1 equivalent) in DCM (10 ml) at room temperature, and the reaction mixture was stirred for 30 min. After the reaction was complete (reaction progress was monitored by LCMS), the volatiles were evaporated under reduced pressure and co-distilled with acetonitrile (3 × 30 ml), and dried under high vacuum for 30 min. The crude product (2.0 g) was used for the next step in the reaction sequence without further purification. LCMS: C 35 H 60 IN3O 12 m / z: 841.32, measured value m / z = 842.32 [M+H] + .

[0156] Program 61: N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)lysine 2,5-dioxopyrrolidine-1-yl ester (8): EDC·HCl was added to a stirred solution of compound 8a (2.0 g, 1 equivalent) and NHS (1.3 equivalent) in DCM (10 ml) at 0 °C, and the mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with 20 ml of DCM, washed with 50 ml of water, dried over Na2SO4, and evaporated. The residue was washed with diethyl ether (2 × 25 ml) to give compound 8 (2.0 g) as a grayish-white solid. LCMS: C 30 H 35 N3O8: m / z: 565.24, measured value m / z = 588.19 [M+Na] + .

[0157] Program 62: N2-(10-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonazo-5,12-diazanechosane-39-acyl)-N6-(4-(4-iodophenyl)butyryl)lysine(9): Add DIPEA (3.0 equivalents) to a solution of 7 (2.0 g, 1 equivalent) in DMF (10 ml), then add a solution of 8 (1.0 equivalent) in DMF at room temperature and stir for 30 minutes. After the reaction is complete (reaction progress monitored by LCMS), evaporate the solvent and purify the crude product by silica gel column chromatography, eluting in MeOH:DCM (0–40%) to give compound 9 (1.7 g), a light brown viscous oil. LCMS: C 61 H 90 IN5O 17 m / z: 1291.54, measured value m / z = 647.46 [M / 2+H] + Program 63: N2-(10-amino-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonazo-5,12-diazanechosane-39-acyl)-N6-(4-(4-iodophenyl)butyryl)lysine(10): Diethylamine (4 ml) was added to a stirred solution of 9 (0.4 g, 1 equivalent) in DCM (4 ml) at room temperature, and the reaction mixture was stirred for 30 min. After the reaction was complete (the reaction progress was monitored by LCMS), the solvent was evaporated under reduced pressure and co-distilled with acetonitrile (3 × 30 ml), and dried under high vacuum for 30 min. Crude product 10 (0.5 g) was used as is in the next step without further purification. LCMS: C 46 H 80 IN5O 15 m / z: 1069.47, measured value m / z = 535.98 [M / 2+H] + .

[0158] Program 64: N2-(10-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azahexadecane-16-amido)-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonoxa-5,12-diazanechos-39-acyl)-N6-(4-(4-iodophenyl)butyryl)lysine(12): DIPEA (3.0 equivalents) was added to a solution of compound 10 (0.5 g, crude, 1 equivalent) in DMF (5 ml), followed by the addition of DMF (5 ml) containing compound 11 (1.0 equivalent) at room temperature, and the reaction mixture was stirred for 30 minutes. After the reaction was complete (monitored by LCMS), the solvent was evaporated. The crude product was purified by silica gel column chromatography (MeOH:DCM, 0-30%) to give compound 9 (0.7 g, crude) as a light brown, viscous oil. LCMS: C 70 H 107 IN6O 21 m / z: 1494.65, measured value m / z = 499.24 [M / 3+H] + 521.5 [M / 3 + Na] + .

[0159] Program 65: N2-(30-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azahexadecane-16-amido)-34-amino-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatritetracoacyl)-N6-(4-(4-iodophenyl)butyryl)lysine(13): TFA (6 ml) was added to a stirred solution of 12 (0.7 g, crude product, 1 equivalent) in DCM (4 ml) at room temperature, and the mixture was stirred for 30 min. After the reaction was complete (reaction progress was monitored by LCMS), the solvent was evaporated under reduced pressure, co-distilled with acetonitrile (3 × 10 ml), and dried under high vacuum for 30 min. The resulting crude product (0.6 g) was used as is in the next step without further purification. LCMS: C 65 H 99 IN6O 19 m / z: 1394.65, measured value m / z = 698.85 [M / 2+H] + . Program 66: 2,2',2''-(10-(8-(1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13-tetraoxa-4-azahexadecane-16-amido)-39-carboxyl-48-(4-iodophenyl)-2,9,37,45-tetraoxa-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraaza-tetraoctadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (15): DIPEA (2 equivalents) was added to solution 13 (0.6 g, crude substance, 1 equivalent) in DMF (5 ml) with stirring. Then, DOTA NHS ester (14, 1 equivalent) was added, and the reaction mixture was stirred for 30 minutes. After the reaction was complete (monitored by LCMS), the solvent was evaporated, and the resulting crude residue (0.65 g, crude substance) was used in its separated state for the next step without further purification. LCMS: C 81 H 125 IN 10 O 26 m / z: 1780.78, measured value m / z = 892.12 [M / 2+H] + .

[0160] Program 67: 2,2',2''-(10-(8-(3-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propamido)-39-carboxyl-48-(4-iodophenyl)-2,9,37,45-tetraoxo-13,16,19,22,25,28,31,34-octaoxo-3,10,38,44-tetraaza-tetraoctadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (16): 15 (0.65 g) was added to diethylamine (5 ml) at room temperature, and the reaction mixture was stirred at the same temperature for 30 min. After completion (reaction progress monitored by LCMS), the solvent was evaporated, and the crude product was purified by preparative HPLC (ACN:H₂O, 5-95%, 40 min) to give 16 (240 mg) as a colorless solid. LCMS: C 66 H 115 IN 10 O 24 m / z: 1558.71, measured value m / z = 780.46 [M / 2+H] + .

[0161] Program 68: 2,2',2''-(10-(39-carboxy-48-(4-iodophenyl)-2,9,37,45-tetraoxo-8-(2-oxo-1-((4-((1-oxo-1-((R)-2-((3aR,4R,6R,7aS)-5,5,7a-trimethylhexahydro-4,6-methylenebenzo[d][1,3,2]dioxoborhexacyclopentane-2-yl) Pyrrolidine-1-yl)prop-2-yl)carbamoyl)quinoline-7-yl)oxy)-6,9,12-trioxa-3-azapentadecan-15-amido)-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazaoctanetetraalkyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (18): Add DIPEA (2 equivalents) to a stirred solution of compound 16 (20 mg, 1 equivalent) in DMF (5 ml), then add compound 17 (1.5 equivalents), and stir the reaction mixture for 30 min. After the reaction is complete (reaction progress monitored by LCMS), evaporate the solvent, and purify the crude residue by preparative HPLC (ACN:H2O, 5-95%, 40 min) to give pure compound 18 (10 mg) as a white solid. LCMS: C 66 H 115 IN 10 O 24 m / z: 2089.97, measured value m / z = 698.31 [M / 3+H] + .

[0162] Program 69: 2,2',2''-(10-(8-(1-((4-((1-((R)-2-boronpyrrolidine-1-yl)-1-oxopropyl-2-yl)carbamoyl)quinoline-7-yl)oxy)-2-oxo-6,9,12-trioxa-3-azapentadecan-15-amido)-39-carboxyl-48-(4-iodophenyl)-2,9,37,45-tetraoxa-13,16,19,22,25,28,31,34-octaoxa-3,10,38,44-tetraazatoctadecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (1411R): To a stirred solution of compound 16 (10 mg, 1 equivalent) in acetone (1 ml), 1 N HCl (1 ml) was added, followed by the addition of methylboric acid (5 equivalents), and the reaction mixture was stirred at room temperature for 12 h. After the reaction was complete (monitored by LC-MS), the compound was purified by preparative HPLC (ACN:H₂O, 5-95%, 40 min (60% within 30 min, then 95% within 40 min)) to obtain a pure compound (4 mg) as a white solid. LCMS: C 85 H 135 BIN 13 O 30 m / z: 1955.86, measured value m / z = 647.31 [M-18 / 3+H] + .

[0163] Synthetic RTX-1409R Following a similar procedure to RTX-1411R, Boc-L-Lys(H)-OtBu was used as the starting material for preparation, but with the following modifications: In procedure 58, 2-(4-iodophenyl)acetic acid is used instead of iodophenylbutyric acid.

[0164] Synthetic RTX-1410R Following a procedure similar to that used for RTX-1411R, Boc-L-Lys(H)-OtBu was used as the starting material for preparation.

[0165] Synthetic RTX-1413R Following a similar procedure to RTX-1411R, Boc-L-Lys(H)-OtBu was used as the starting material for preparation, but with the following modifications: In procedure 58, 2-(4-iodophenyl)acetic acid is used instead of iodophenylbutyric acid.

[0166] Use step 23 instead of procedure 66 to install the Macropa chelating agent.

[0167] Synthetic RTX-1414R The preparation was carried out using a procedure similar to that of RTX-1411R, with Boc-L-Lys(H)-OtBu as the starting material, but with the following modifications: Use step 23 instead of step 66 to install the Macropa chelating agent.

[0168] Synthetic RTX-1415R It is prepared using a procedure similar to that of RTX-1411R, but with the following modifications: Use procedure 23 instead of procedure 66 to install Macropa chelating agent.

[0169] Example 2 - The compounds of this invention bind to FAP with high affinity. As described below, the protease reaction was assembled in a 384-well plate (Greiner) in a total volume of 20 μL.

[0170] The recombinant protein was pre-diluted in assay buffer containing 100 mM HEPES, pH 7.5, 0.1% BSA, 0.01% Triton X-100, and 1 mM DTT, and then dispensed into 384-well plates (10 μL / well). The test compound was serially pre-diluted in DMSO and added to the wells by sonication (Labcyte Echo 550). Four replicates were assembled with control samples (0% inhibition in the absence of inhibitor, DMSO only) and 100% inhibitor (in the absence of enzyme) to calculate the % inhibition in the presence of the compound. The concentration of DMSO in all samples was 1%.

[0171] The compound was pre-incubated with the enzyme for 15 minutes. Human FAP was obtained from Enzo, catalog number BML-SE409-0010. The reaction was initiated by adding 10 μL of 2x FAM-labeled substrate peptide (FAM-GPRPFNYLAKK-NH2) prepared in the same assay buffer. The final enzyme concentration was 0.5 nM. The final substrate peptide concentration was 1 μM.

[0172] The reaction was carried out at room temperature. Incubation time for human FAP was three hours, and for mouse FAP, it was 0.5 hours. After incubation, the kinase reaction was quenched by adding 50 μL of stop buffer: at 100 x IC50. 50 Supplement the assay buffer for the reference inhibitor.

[0173] The terminated plate was analyzed using a microfluidic electrophoresis system (Caliper LabChip® 3000, Caliper Life Sciences / PerkinElmer). The relative intensity change of the peptide substrate and lysis products was the measured parameter. The activity in each test sample was determined as the product-to-total ratio (PSR): P / (S+P), where P is the peak height of the product and S is the peak height of the substrate. The percentage of inhibition (P0) was determined using the following equation. inh ):Pinh = (PSR 0%inh - PSR 化合物 ) / (PSR 0%inh - PSR 100%inh ) 100, of which: PSR 化合物 PSR is the ratio of product to total in the presence of the compound. 0%inh The product / total ratio and PSR in the absence of compounds. 100%inh This is the ratio of product to total in the absence of an enzyme. It is used to determine the IC50 of a compound. 50 (50% inhibition), the %-inh data (P) were fitted using XLfit software (IDBS) with a 4-parameter sigmoid dose-response model. inh (Compare compound concentrations). These values ​​are summarized in Table 1 and grouped, where AA represents IC50. 50 <250 pM, A indicates IC 50 <0.1nM; B is IC 50 From 0.1 to 0.5 nM; C is IC 50 From 0.5 to 5.0 nM; D is IC 50 From 5.0 to 100 nM; E is IC 50 >100 nM. IC50 values ​​of some compounds in this disclosure. 50 The value exceeded the detection limit of the assay (i.e., below 250 pM).

[0174] Table 1: Results of the affinity study

[0175] The S stereoisomer of pyrrolidine with the S configuration is much more potent than the corresponding R stereoisomer. The aforementioned inhibition studies also indicate that prolongation... (It has an LC of 1.5 nM) 50 The activity is significantly improved by extending the quinoline core chain. Specifically, each chain elongation brings improvement, and the addition of a terminal amino acid (lysine) provides further enhancement of activity.

[0176] In the surface plasmon resonance measurement data, a greater difference was observed between some of the compounds of the present invention and FAPI-46 (the affinity of FAPI-46 was measured to be 255 pM, while the affinity of some of the disclosed compounds was 20 pM or stronger).

[0177] Example 3 – Surface plasmon resonance studies on the binding affinity of the compounds of this invention As described in Example 2, some compounds of the present invention exhibited binding affinity exceeding the detection limit of this assay (i.e., approximately 250 pM). Surface plasmon resonance (SPR) provides a significantly lower detection limit of approximately 1–10 pM. SPR assays were performed on the unmetallized and non-radioactive lanthanum (La) and lutetium (Lu) labeled compounds of the present invention to analyze their binding affinity for human, mouse, and rat FAP.

[0178] SPR method: SPR experiments were performed at 37 °C using a Biacore 8K (Cytiva Europe GmbH). For all eight channels, ligands were immobilized on an SA chip (Cytiva #BR100531) via SA-biotin capture (mouse FAP, Biosystems Acro / catalog number FAP-M82Q8; human FAP, Biosystems Acro / catalog number FAP-H82Q6) or on a CM5 chip (Cytiva #29149603) in flow cell 2 via amide coupling (rat FAP, Biosystems Acro / catalog number FAP-R5246). For immobilization, ligand solutions at concentrations of 10–20 μg / mL were prepared and run on the surface of the selected flow cell at a flow rate of 5 μL / min using DMSO-free running buffer HBS-P+ (Cytiva #BR100671). The flow cell 1 of each of the 8 channels is left empty as a reference surface.

[0179] K of the compound D The assays were performed in a single-cycle experiment. HBS-P+ buffer containing 1% DMSO was used as the run buffer. Six concentrations (100 nM, 25 nM, 6.25 nM, 1.56125 nM, 0.390625 nM, 0.0976525 nM or 400 nM, 100 nM, 25 nM, 6.25 nM, 1.56125 nM, 0.390625 nM) and a zero value were used as blanks in a 1:4 dilution series. The experiments were conducted at a flow rate of 100 μL / min, with an contact time of 80 seconds for each solution and a final dissociation time of 2500 seconds. Solvent correction was performed at the beginning and end of the experiments.

[0180] All data were analyzed using Biacore Insight evaluation software (version 5.0.18.22102 Cytiva 2022). Flow cell 1 was used as a reference in all channels, and all experiments were solvent corrected using the measured calibration curves. Single-cycle data were analyzed using a 1:1 kinetic model. All measurements were repeated two or three times.

[0181] Table 2 below summarizes the average dissociation constant (K) determined by n=2 or 3 repeated experiments. D The binding affinity of some compounds of this invention exceeds the detection limit of this assay (i.e., <1 pM). Labelling with non-radioactive La or Lu did not significantly alter the binding affinity of FAP for the tested compounds, as assessed within the limits of the SPR assay.

[0182] Table 2. Results of the SPR combined study

[0183] Example 4 -use 18 Radiolabeling of FAP-targeted compounds using the F direct labeling method Received from the manufacturer 18 F was loaded onto a Sep Pak QMA Light Plus column and eluted with Cs2CO3 / K222 solution into a glass reaction vial. The eluted sample was then... 18 F was azeotropically dried at 95°C under a nitrogen stream using acetonitrile (3 × 1 ml). 2.5 mg of the targeted FAP compound was dissolved in 0.3 ml DMSO and then added to the dried solution. 18 The above-mentioned vial of F was filled. The vial was sealed and heated to 90°C for 15 minutes. The vial was cooled and 1 N HCl solution was added, then it was sealed and heated to 95°C for 10 minutes. The reaction mixture was cooled and neutralized, then loaded onto a SemiPrep HPLC column for purification and prepared appropriately for use. Alternatively, a suitable FAP-targeting compound with a chelating agent can be used. 18 F AlF labeling, wherein an AlCl3 reserve (22.5 µL, 45 nmol, 0.9 equivalents) in acetate buffer is added to sodium acetate. 18 Add 200 μL of solution F and incubate the reaction vial at room temperature for 5 min. Then add 12.5 µL of the targeted FAP compound solution (50 nmol scale) from the precursor stock to the above vial. Adjust the pH to approximately 4.0 by adding 1% v / v aqueous acetic acid (15 µL). Add 200 µL of EtOH co-solvent and seal the reaction vial, then heat at 100 °C for 15 min. Dilute the reaction mixture to 9.5 mL and load it onto a C18 Sep Pak column. Elute the product with 300 μL of acidified EtOH and prepare for use.

[0184] Example 5 – Radiolabeling methods and results General procedure: Combine 20 µg of the precursor (unless otherwise specified) with the indicated radioisotope. The radioisotope in the HCl solution is obtained from the generator (used for... 68 The eluted solution (Ga) or commercially available HCl solution was buffered with varying amounts of 3NNaOAc to obtain a final pH of 4–6. Reactions were performed using C18 Sep-Pak Lite columns as needed to obtain additional purity and / or reformulation for injection. Labeling results are shown in Table 3.

[0185] Table 3. Results of representative labeled reactions

[0186] Example 6 – Detection of tumors in a rodent model using compounds of the present invention Approximately 10 lb / c nude mice were transplanted 7 Inducing the growth of U-87 (human glioblastoma) cells and promoting tumor development. A certain amount of the present invention was administered to mice via intravenous injection (e.g., tail vein). 18 Mice were euthanized one hour after receiving an F-labeled FAP-targeting agent. Imaging was then performed on the mice to evaluate their efficacy. 18 The binding of F-labeled FAP-targeting compounds to tumors was assessed. The biodistribution of the compounds was evaluated by analyzing tissue samples removed from various organs.

[0187] Example 7 –Biodistribution research U87MG cells were subcutaneously implanted into the right shoulder of female BALB / c nude mice in a 1:1 matrigel:PBS solution. When the tumor reached a volume of 150–500 mm³, the radiolabeled ligand was administered intravenously (IV) via the tail vein. Mice were humanely euthanized by exsanguination at various time points post-injection, and tissue samples (bladder, blood, urine, bone (femur), heart, lungs, liver, both kidneys, small intestine (including contents), large intestine (including contents), muscle (quadriceps), tumor, and tail) were removed, weighed, and counted using a gamma counter. Activity of each collected tissue was measured in counts per minute (CPM). Tripartite samples of the radiotracer were also measured in the gamma counter to calculate the coefficient (µCi / CPM) for converting the counts to activity units. Decay values ​​were corrected for injection time and for background radiation. The biodistribution of compounds [177Lu]RTX-1371R, [177Lu]RTX-1391R, and [177Lu]RTX-1392R shows their localization within tumors and their lowest concentrations in other organs (see [177Lu]RTX-1371R, [177Lu]RTX-1391R, [177Lu]RTX-1392R). Figure 1-3This biodistribution indicates increased tumor selectivity over time, demonstrating a high affinity for FAP. Incorporation of D-alanine significantly increased tumor retention rates at both 72 and 168 hours (see [link to relevant documentation]). Figure 4-6 Compared with existing compounds [177Lu]PNT5555 ([177Lu]RTX-1418R) and [177Lu]3BP-2286 ([177Lu]RTX-1386S), the compounds disclosed herein exhibit significantly higher tumor retention rates at late time points (48–168 hours post-injection) (see [link to original text]). Figure 7 and Figure 8 ).

[0188] Example 8 - Cryo-fluorescence computed tomography (CFT) experiment Female Nu / J or NCr-Foxn1 nude mice were subcutaneously inoculated with U-87MG cells in a 1:1 matrix gel:PBS solution in the right shoulder. Tumors were inoculated when the tumor volume reached at least 150 mm². 3 1.5 µg of the ligand dissolved in sterile PBS was administered intravenously (IV) via tail injection. Mice were euthanized at different time points post-injection via CO2 inhalation and cervical dislocation, and then frozen in a hexane bath for 10 minutes, the hexane bath being cooled with dry ice around a hexane-filled metal container for 30 minutes before freezing. Mice were stored at -80°C until embedded in frozen OCT compound blocks, and the distribution of the ligand in the mice was imaged in Xerra (Emit Imaging) using cryo-fluorescence tomography (CFT). A 35 µm thick slice was cut from the top of the sample block using a razor blade, followed by fluorescence excitation, and then the fluorescence emission spectrum was read at the corresponding wavelength of the fluorophore coupled to the ligand for 500 ms. In addition to the fluorescence images, RGB images were acquired after each slice. Slicing and image acquisition were performed throughout the tissue block. Data were processed using Xerra Recon software (Emit Imaging) and analyzed using Vivoquant (Invicro). A single spherical region of interest (ROI) was drawn within the organ of interest for the heart, liver, lungs (one ROI per organ), kidneys (one ROI per kidney), joints (right posterior knee), muscles (right posterior quadruped), and brain. Regions of interest (ROIs) throughout the body were also drawn. Two-dimensional ROIs were drawn within the entire tumor, and then interpolated to form a three-dimensional ROI covering the entire tumor volume. The mean fluorescence values ​​(in arbitrary units) of the ROIs in mice injected with different compounds at different time points were compared, see [link to relevant documentation]. Figure 10 The mean organ ROI is standardized to the whole-body or muscle ROI value by dividing the ROI values ​​of all other organs by the whole-body or muscle ROI value. See [link to documentation]. Figure 11The average of these standardized ROIs was compared for different compounds and time points to assess the signal-to-noise ratio.

[0189] Example 9 – Efficacy and tolerability study of U-87 xenografts Female Nu / J mice were subcutaneously inoculated with U-87MG cells in a 1:1 matrix gel:PBS mixture on the right ventral side. Tumor volume (using formula 1 / 2 × length) was measured. 2 (width) reaches 120 mm 3 Up to 250 mm 3 Between treatments, mice were weighed and randomly assigned to treatment groups based on tumor volume. [The remaining text appears to be incomplete and requires further context.] 225 The treatment group received a single intravenous (IV) tail injection of 1 µg of ligand with an activity of 15, 30, or 60 kBq. The multi-dose group received four IV tail injections of 1 µg ligand with an activity of 15 kBq (2–3 days apart) every two weeks, for a cumulative dose of 60 kBq. Body weight and tumor volume were recorded twice weekly. If a mouse lost more than 10% of its body weight at the start of the study, DietGel® Boost (ClearH2O) was administered to all cages in the study. Tumor volume, body weight, and survival were plotted using GraphPad Prism software; see [link to relevant documentation]. Figure 12-17 Measurements were performed on each mouse every two weeks until one of the following endpoint criteria was met: a) tumor volume ≥2000 mmHg. 3 b) Body weight decreased by ≥20% from the start of the study; c) Tumors ulcerated; at this point, the mice were euthanized by inhaling CO2 and dislocating their cervical spine.

[0190] equivalent While certain embodiments have been described and illustrated by those skilled in the art, modifications, substitutions of equivalents, and other types of alterations can be made to the compounds of the present technology or their salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers, or racemic mixtures described herein after reading the foregoing specification. Each of the foregoing aspects and embodiments may also include or combine these variations or aspects disclosed with respect to any or all other aspects and embodiments.

[0191] This technology is not limited to the specific aspects described herein, which are intended as a single illustration of various aspects of this technology. As will be apparent to those skilled in the art, many modifications and variations can be made to this technology without departing from its spirit and scope. Functionally equivalent methods within the scope of this technology, in addition to those listed herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. It should be understood that this technology is not limited to specific methods, reagents, compounds, compositions, labeled compounds, or biological systems, although variations are possible. It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting. Therefore, this specification is considered exemplary only within the breadth, scope, and spirit of this technology as indicated by the appended claims, their definitions, and any equivalents.

[0192] All publications, patents and other documents referenced in this specification are incorporated herein by reference in their entirety.

Claims

1. A compound represented by the following structural formula: ； Or its pharmaceutically acceptable salt, wherein: n is 0 or 1; Z represents NH, O, S, and CR. 6 R 7 NHCO, CONH, or 4-7 membered nitrogen-containing heterocycles bonded to Y via cyclic nitrogen atoms of heterocycles; A is NH, O, S, or CR. 6 R 7 ; B comprises a branched, unbranched, or cyclic aliphatic group of up to 30 carbon atoms, optionally interrupted by a peptide chain of up to 10 heteroatoms or up to 20 amino acid residues, wherein B is optionally substituted by 1-5 groups selected from: F, Cl, Br, I, =O, OR 6 OCOR 6 COOR 6 CN, =NR 6 NR 6 R 7 =S and SR 6 The condition is that B contains at least 3 atoms in the chain between group D and group A; D is selected from: OPO3H2, PO3H2, OSO3H, SO3H and COOH or their C1-C4 alkyl esters; X is O or S; R 1 It is a chelating group, an optical dye or fluorophore, a cytotoxic agent or an immunostimulant, or optionally composed of one or more R... 5 The group indicated is the benzoyl group that has been substituted; R 3 It is a C1-C8 alkyl or a C1-C4 aralkyl, wherein: The alkyl and aryl moieties of the alkyl group and the aryl group are each optionally and independently surrounded by F, Cl, Br, I, branched, unbranched, or cyclic C1-C6 aliphatic groups, OR 6 OCOR 6 COOR 6 CHO, COR 6 CH2OR 6 NR 6 R 7 CH2NR 6 R 7 SR 6 =O, =S and =NH substitution; R 4 It is either CN or B(OH)2; Each R 5 Independently selected from halogen, cyano, halomethyl, N + (CH3)3W - W - It is a pharmaceutically acceptable anion; R 6 and R 7 Independently selected from: H or C1-C6 alkyl; and R 8 It is a C1-C4 alkyl group, and R 9 Selected from H and C1-C4 alkyl groups, or R 8 and R 9 Together with its middle carbon atom, it forms a C3-C6 cycloalkyl group.

2. The compound according to claim 1, wherein B is independently a branched or unbranched aliphatic group of 3 to 20 carbon atoms, said carbon atom optionally being interrupted by a peptide chain of up to 10 heteroatoms or up to 5 amino acid residues, said aliphatic group optionally being F, Cl, Br, I, =O, OR 6 OCOR 6 COOR 6 CN, =NR 6 NR 6 R 7 =S or SR 6 replace.

3. The compound according to claim 1 or 2, wherein it is represented by the following structural formula: ; Or its pharmaceutically acceptable salt, wherein m is an integer from 0 to 12; o is 0 or 1; and R 2 It is H or C1-C4 alkyl.

4. The compound according to claim 3, wherein it is represented by the following structural formula: ； Or its pharmaceutically acceptable salt.

5. The compound according to claim 3, wherein the compound is represented by the following structural formula: ； Or its pharmaceutically acceptable salt.

6. The compound according to claim 3 or 4, wherein it is represented by the following structural formula. or its pharmaceutically acceptable salt.

7. The compound according to any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein R 3 It can be optionally reacted with F, Cl, Br, I or C 1-4 Alkyl-substituted C 1-8 Alkyl or C 1-4 Aryl group.

8. The compound according to any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein R 3 It is optional to be I or C 1-4 Alkyl-substituted C 1-8 Alkyl or C 1-4 Aryl group.

9. The compound according to any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein R 3 It is a C1-C8 alkyl or C1-C4 aralkyl that is optionally substituted with a C1-C4 alkyl group.

10. The compound according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof, wherein R 3 It is methyl, (4-isobutylphenyl)methyl, (4-isobutylphenyl)propyl, (4-iodophenyl)methyl or (4-iodophenyl)propyl.

11. The compound according to any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein R 3 It is methyl, (4-isobutylphenyl)methyl and (4-isobutylphenyl)propyl.

12. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein R8 is methyl and R9 is hydrogen.

13. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 12, wherein o is 1 and m is 3 to 12.

14. The compound of claim 13 or a pharmaceutically acceptable salt thereof, wherein m is 8.

15. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 12, wherein o is 0.

16. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15, wherein n is 1.

17. The compound according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein R 1 It is a fluorophore or optical dye.

18. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein the fluorophore is The optical dyes are selected from: carbocyanine, indole carbocyanine, oxycarbocyanine, sulfur carbocyanine, cyanine, polyacetylenes, coumarin, rhodamine, xanthan, fluorescein, boron dipyrrole methylene (BODIPY), VivoTag-680, VivoTag-S750, AlexaFluor dyes (e.g., AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790) and DylightFluor dyes.

19. The compound according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein R 1 It is a chelating group, which is a residue of a chelating agent.

20. The compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein the chelating group is a residue selected from the group consisting of: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), p-SCN-Bn-NOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS), PIP-DOTA, diethylenetriaminepentaacetic acid (DTPA), PIP-DTPA, AZEP-DTPA, ethylenediaminetetraacetic acid (EDTA), triethylenetetramine-N,N,N',N'',N''',N'''-hexadecane Acetic acid (TTHA), 7-[2-(bis(carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraazacyclododecane-1-yl-acetic acid (DEPA), 2,2',2''-(10-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanophenyl)pentyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (3p-C-DEPA-NCS), NETA, {4-carboxymethyl-7-[2-(carboxymethylamino)-ethyl]-perhydro-1,4,7-triazacyclononane-1-yl}-acetic acid (NPTA), diacetylpyridine bis(benzoylhydrazone), 1,4,7,10,13,16 -Hexaazacyclooctadecane N,N',N'',N''',N'''',N''''-Hexaacetic acid (HEHA), octadentate terephthalamide ligand, 2,2'-(4-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanophenyl)pentyl)-10-(2-(bis(carboxymethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid, N,N'-bis[(6-carboxy-2-pyridinyl)methyl]-4,13-diaza-18-crown-6 (H2macropa), 6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7- (16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7-yl)methyl)-4-isothiocyanopyridinic acid (macropa-NCS), 3,9-carboxymethyl-6-(2-methoxy-5-isothiocyanophenyl)carboxymethyl-3,6,9,15-tetraazabicyclo-[9.3.1]pentadecane-1(15),11,13-triene and 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetamide (TCMC or DOTAM).

21. The compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein the residue of the chelating agent is a macropa-NCS or macropa-NCO residue.

22. The compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein the residue of the chelating agent is a residue of p-SCN-Bn-NOTA, p-SCN-Bn-DOTA, NOA, or DOTA.

23. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-16 or 19, wherein the chelating group is a ferrophilic residue.

24. The compound according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein: R 1 It is optionally controlled by one or more R 5 The group indicated is the benzoyl group that has been substituted; Each R 5 Independently selected from halogen, cyano, halomethyl, N + (CH3)3W - ;and W - It is a pharmaceutically acceptable anion.

25. The compound of claim 24 or a pharmaceutically acceptable salt thereof, wherein each R 5 Independently selected from fluorine, cyano, trifluoromethyl, N + (CH3)3W - .

26. The compound according to claim 24 or 25, or a pharmaceutically acceptable salt thereof, wherein R 5 The halogen or fluorine group represented is 18 F.

27. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-26, wherein R 2 It is H and R 4 It is B(OH)2.

28. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-26, wherein R 2 It is H and R 4 It's CN.

29. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, represented by a structural formula selected from the following: ； ； ； ； ； ； ;and .

30. The compound of claim 1 or a pharmaceutically acceptable salt thereof, represented by a structural formula selected from the following: 。 31. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, 19-23 or 27-30, wherein the residues of the chelating agent are chelated with a radionuclide.

32. The compound of claim 31 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is selected from... 177 Lu、 175 Lu、 45 Sc、 64 Cu、 67 Cu、 68 Cu、 66 Ga、 67 Ga、 68 Ga、 69 Ga、 71 Ga、 90 Y、 89 Y、 86 Y、 89 Zr、 90 Y、 99m Tc, 111 In、 113 In、 115 In、 139 La、 134 Ce、 136 Ce、 138 Ce、 140 Ce、 142 Ce、 151 Eu、 153 Eu、 152 Dy、 149 Tb, 159 Tb, 154 Gd, 155 Gd, 156 Gd, 157 Gd, 158 Gd, 160 Gd, 188 Re、 186 Re、 213 Bi、 211 At、 217 At、 227 Th、 226 Th、 225 Ac、 233 Ra、 152 Dy、 213 Bi、 212 Bi、 211 Bi、 203 Pb, 212 Pb, 255 Fm and uranium-230.

33. The compound of claim 31 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is an alpha-emitting radionuclide, such as... 225 Ac、 233 Ra and 212 Pb.

34. The compound of claim 31 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is an Auger electron-emitting radionuclide or a beta-emitting radionuclide, such as... 177 Lu、 90 Y and 67 Cu.

35. The compound of claim 21 or a pharmaceutically acceptable salt thereof, wherein the residues of macropa-NCS or macropa-NCO are associated with... 225 Ac is chelated.

36. A pharmaceutical composition comprising: a. The compound of any one of claims 1-35 or a pharmaceutically acceptable salt thereof; and b. Pharmaceutically acceptable carriers or diluents.

37. A method of treating diseased tissue in a subject, wherein the diseased tissue expresses fibroblast activation protein α, the method comprising administering to the subject an effective amount of the compound or pharmaceutically acceptable salt of any one of claims 31-35 or the pharmaceutical composition of claim 36, and wherein the radionuclide is a therapeutic radionuclide.

38. The method of claim 37, wherein the diseased tissue is cancer.

39. The method of claim 38, wherein the cancer is pancreatic cancer, liver cancer, gallbladder cancer, neuroblastoma, breast cancer, ovarian cancer, esophageal cancer, kidney cancer, prostate cancer, colorectal cancer, soft tissue sarcoma, osteosarcoma, or melanoma.

40. The method of claim 38, wherein the diseased tissue is fibrotic.

41. A method for imaging a region in a subject having or suspected of having diseased or fibrotic tissue expressing fibroblast activation protein α, comprising: a. Administering to the subject a diagnostically effective amount of any one of claims 16-19, 25-30, or 32-35, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 36, wherein the radionuclide is a diagnostic radionuclide; b. Expose the area in the subject to the imaging device; and c. Obtain an image of the diseased tissue in the region.

42. The method of claim 41, wherein the region has or is suspected to have diseased tissue including primary cancer or metastatic cancer.

43. The method of claim 41, wherein the region has or is suspected to have a diseased area comprising fibrotic tissue.

44. A method for imaging a tumor, the method comprising: a. To contact the tumor and / or surrounding tissue with the compound of any one of claims 18-19 or a pharmaceutically acceptable salt thereof in an amount sufficient to bind to the tumor; b. Irradiate the tumor and / or surrounding tissue at a wavelength absorbed by the compound; c. and detecting signals from the compound to image the tumor and / or surrounding tissue.

45. A method for treating diseased tissue, comprising: a. Administering to a subject any one of claims 18-19, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in an amount that effectively contacts and binds to the diseased tissue; b. Using the compound as a reference, irradiate the area bound to the compound with one or more doses of external irradiation, thereby treating the diseased tissue with irradiation.

46. ​​The method of claim 45, wherein the compound comprises a radionuclide emitting gamma rays or positrons, or an optical dye or fluorophore, or other chelating group capable of detecting irradiation.

47. A method for treating diseased tissue, comprising: The compound of any one of claims 18-19, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, is administered to the subject in an amount that effectively contacts and binds to the diseased tissue; and the compound is used as a reference for guiding surgical application to excise the area of ​​the diseased tissue, thereby excising the diseased tissue.

48. The method of claim 47, wherein the compound comprises a radionuclide emitting gamma rays or positrons, or an optical dye or fluorophore, or other chelating group capable of detecting irradiation.

49. The method of claim 37, further comprising administering a chemotherapeutic agent, an immunotherapeutic agent, or irradiating the diseased tissue with one or more doses of external irradiation.

50. The method of claim 38, wherein the compound or a pharmaceutically acceptable saline thereof is administered intravenously, subcutaneously, intramuscularly, locally, or directly to the bladder to a subject in need.

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